Method For Producing Unsaturated Omega-3-Fatty Acids In Transgenic Organisms

ABSTRACT

The present invention relates to nucleic acid sequences coding for polypeptides with ω-3-desaturase activity. The invention furthermore relates to nucleic acid constructs, vectors and organisms comprising at least one nucleic acid sequence according to the invention, at least one vector comprising the nucleic acid sequence and/or the nucleic acid constructs, and transgenic organisms comprise the abovementioned nucleic acid sequences, nucleic acid constructs and/or vectors.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/590,958, filed Aug. 25, 2006, which is the national stage application(under 35 U.S.C. 371) of PCT/EP2005/001865 filed Feb. 23, 2005, whichclaims benefit of German application 10 2004 009 458.6 filed Feb. 27,2004. The entire contents of each of these applications are herebyincorporated by reference herein.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is Sequence_Listing_(—)13987_(—)00118. The size ofthe text file is 10 KB, and the text file was created on Apr. 27, 2010.

FIELD OF THE INVENTION

The present invention relates to a process for production of unsaturatedω-3-fatty acids and to a process for production of triglycerides with anelevated content of unsaturated fatty acids, especially of ω-3-fattyacids having more than three double bonds. The invention relates to theproduction of a transgenic organism, preferably of a transgenic plant orof a transgenic microorganism, with an elevated content of unsaturatedω-3-fatty acids, oils or lipids having ω-3-double bonds as the result ofthe expression of an ω-3-desaturase from fungi of the family Pythiaceaesuch as the genus Phytophtora, for example of the genus and speciesPhytophtora infestans.

DESCRIPTION OF RELATED ART

The invention furthermore relates to the nucleic acid sequences, nucleicacid constructs, vectors and organisms comprising at least one nucleicacid sequence according to the invention, at least one vector comprisingthe nucleic acid sequence and/or the nucleic acid constructs, andtransgenic organisms comprise the abovementioned nucleic acid sequences,nucleic acid constructs and/or vectors.

A further part of the invention relates to oils, lipids and/or fattyacids produced by the process according to the invention, and to theiruse. The invention moreover relates to unsaturated fatty acids andtriglycerides with an elevated content of unsaturated fatty acids andtheir use.

Fatty acids and triacylglycerides have a multiplicity of applications inthe food industry, in animal nutrition, in cosmetics and thepharmacological sector. Depending on whether they are free saturated orunsaturated fatty acids or else triacylglycerides with an elevatedcontent of saturated or unsaturated fatty acids, they are suitable forvery different applications.

Polyunsaturated long-chain ω-3-fatty acids such as eicosapentaenoic acid(=EPA, C20:5^(Δ5,8,11,14,17)) or docosahexaenoic acid (=DHA,C22:6^(Δ4,7,10,13,16,19)) are important components of human nutritionowing to their various roles in health aspects, including thedevelopment of the child brain, the functionality of the eyes, thesynthesis of hormones and other signal substances, and the prevention ofcardiovascular disorders, cancer and diabetes (Poulos, A Lipids 30:1-14,1995; Horrocks, L A and Yeo Y K Pharmacol Res 40:211-225, 1999). Thereis therefore a demand for the production of polyunsaturated long-chainfatty acids.

Owing to the present-day composition of human food, an addition ofpolyunsaturated ω-3-fatty acids, which are preferentially found in fishoils, to the food is particularly important. Thus, for example,polyunsaturated fatty acids such as DHA or EPA are added to infantformula to improve the nutritional value. The unsaturated fatty acid DHAis supposed to have a positive effect on the development and maintenanceof brain functions.

In the text which follows, polyunsaturated fatty acids are referred toas PUFA, PUFAs, LCPUFA or LCPUFAs (poly unsaturated fatty acids, PUFA,long chain poly unsaturated fatty acids, LCPUFA).

The various fatty acids and triglycerides are mainly obtained frommicroorganisms such as Mortierella or Schizochytrium or fromoil-producing plants such as soybeans, oilseed rape, algae such asCrypthecodinium or Phaeodactylum and others, being obtained, as a rule,in the form of their triacylglycerides (=triglycerides=triglycerols).However, they can also be obtained from animals, for example, fish. Thefree fatty acids are advantageously prepared by hydrolysis. Verylong-chain polyunsaturated fatty acids such as DHA, EPA, arachidonicacid (=ARA, C20:4^(Δ5,8,11,14)), dihomo-γ-linolenic acid(C20:3^(Δ8,11,14)) or docosapentaenoic acid (DPA,C22:5^(Δ7,10,13,16,19)) are not synthesized in plants, for example inoil crops such as oilseed rape, soybeans, sunflowers and safflower.Conventional natural sources of these fatty acids are fish such asherring, salmon, sardine, redfish, eel, carp, trout, halibut, mackerel,zander or tuna, or algae.

Depending on the intended use, oils with saturated or unsaturated fattyacids are preferred. In human nutrition, for example, lipids withunsaturated fatty acids, specifically polyunsaturated fatty acids, arepreferred. The polyunsaturated ω-3-fatty acids are said to have apositive effect on the cholesterol level in the blood and thus on thepossibility of preventing heart disease. The risk of heart disease,stroke or hypertension can be reduced markedly by adding these ω-3-fattyacids to the food. Also, ω-3-fatty acids have a positive effect oninflammatory, specifically on chronically inflammatory, processes inassociation with immunological diseases such as rheumatoid arthritis.They are therefore added to foodstuffs, specifically to dieteticfoodstuffs, or are employed in medicaments. ω-6-fatty acids such asarachidonic acid tend to have an adverse effect on these disorders inconnection with these rheumatic diseases on account of our usual dietaryintake.

ω-3- and ω-6-fatty acids are precursors of tissue hormones, known aseicosanoids, such as the prostaglandins, which are derived fromdihomo-γ-linolenic acid, arachidonic acid and eicosapentaenoic acid, andof the thromoxanes and leukotrienes, which are derived from arachidonicacid and eicosapentaenoic acid. Eicosanoids (known as the PG₂ series)which are formed from the ω-6-fatty acids generally promote inflammatoryreactions, while eicosanoids (known as the PG₃ series) from ω-3-fattyacids have little or no proinflammatory effect. Therefore food having ahigh proportion of ω-3-fatty acid has a positive effect on human health.

Owing to their positive characteristics, there has been no lack ofattempts in the past to make available genes which are involved in thesynthesis of fatty acids or triglycerides for the production of oils invarious organisms with a modified content of unsaturated fatty acids.Thus, WO 91/13972 and its US equivalent describe a Δ9-desaturase. WO93/11245 claims a Δ15-desaturase and WO 94/11516 a Δ12-desaturase.Further desaturases are described, for example, in EP-A-0 550 162, WO94/18337, WO 97/30582, WO 97/21340, WO 95/18222, EP-A-0 794 250, Stukeyet al., J. Biol. Chem., 265, 1990: 20144-20149, Wada et al., Nature 347,1990: 200-203 or Huang et al., Lipids 34, 1999: 649-659. However, thebiochemical characterization of the various desaturases has beeninsufficient to date since the enzymes, being membrane-bound proteins,present great difficulty in their isolation and characterization (McKeonet al., Methods in Enzymol. 71, 1981: 12141-12147, Wang et al., PlantPhysiol. Biochem., 26, 1988: 777-792). As a rule, membrane-bounddesaturases are characterized by being introduced into a suitableorganism which is subsequently analyzed for enzyme activity by analyzingthe starting materials and the products. Δ6-Desaturases are described inWO 93/06712, U.S. Pat. No. 5,614,393, U.S. Pat. No. 5,614,393 WO96/21022, WO 00/21557 and WO 99/27111, and also the application for theproduction in transgenic organisms is described in WO 98/46763, WO98/46764 and WO 98/46765. Here, the expression of various desaturases isalso described and claimed in WO 99/64616 or WO 98/46776, as is theformation of polyunsaturated fatty acids. As regards the expressionefficacy of desaturases and its effect on the formation ofpolyunsaturated fatty acids, it must be noted that the expression of asingle desaturase as described to date has only resulted in low contentsof unsaturated fatty acids/lipids such as, for example, γ-linolenic acidand stearidonic acid. Furthermore, mixtures of ω-3- and ω-6-fatty acidsare usually obtained.

Especially suitable microorganisms for the production of PUFAs aremicroorganisms such as microalgae such as Phaeodactylum tricornutum,Porphiridium species, Thraustochytrium species, Schizochytrium speciesor Crypthecodinium species, ciliates such as Stylonychia or Colpidium,fungi such as Mortierella, Entomophthora or Mucor and/or mosses such asPhyscomitrella, Ceratodon and Marchantia (R. Vazhappilly & F. Chen(1998) Botanica Marina 41: 553-558; K. Totani & K. Oba (1987) Lipids 22:1060-1062; M. Akimoto et al. (1998) Appl. Biochemistry and Biotechnology73: 269-278). Strain selection has resulted in the development of anumber of mutant strains of the microorganisms in question which producea series of desirable compounds including PUFAs. However, the mutationand selection of strains with an improved production of a particularmolecule such as the polyunsaturated fatty acids is a time-consuming anddifficult process, which is why as described above recombinant methodsare preferred wherever possible. However, only limited amounts of thedesired polyunsaturated fatty acids such as DPA, EPA or ARA can beproduced with the aid of the abovementioned microorganisms; where theyare generally obtained as fatty acid mixtures of, for example, EPA, DPAand ARA, depending on the microorganism used.

A variety of synthetic pathways is being discussed for the synthesis ofthe polyunsaturated fatty acids arachidonic acid, eicosapentaenoic acidand docosahexaenoic acid (FIG. 1). Thus, EPA or DHA are produced innumerous marine bacteria such as Vibrio sp. or Shewanella sp. via thepolyketide pathway (Yu, R. et al. Lipids 35:1061-1064, 2000; Takeyama,H. et al. Microbiology 143:2725-2731, 1197)).

An alternative strategy is the alternating activity of desaturases andelongases (Zank, T. K. et al. Plant Journal 31:255-268, 2002;Sakuradani, E. et al. Gene 238:445-453, 1999). A modification of theabove-described pathway by Δ6-desaturase, Δ6-elongase, Δ5-desaturase,Δ5-elongase and M-desaturase is the Sprecher pathway (Sprecher 2000,Biochim. Biophys. Acta 1486:219-231) in mammals. Instead of theΔ4-desaturation, a further elongation step is effected here to give C₂₄,followed by a further Δ6-desaturation and finally β-oxidation to givethe C₂₂ chain length. What is known as the Sprecher pathway (see FIG. 1)is, however, not suitable for the production in plants andmicroorganisms since the regulatory mechanisms are not known.

Depending on their desaturation pattern, the polyunsaturated fatty acidscan be divided into two large classes, viz. ω-6- or ω-3-fatty acids,which differ with regard to their metabolic and functional activities(FIG. 1).

The starting material for the ω-6-metabolic pathway is the fatty acidlinoleic acid (18:2^(Δ9,12)) while the ω-3-pathway proceeds vialinolenic acid (18:3^(Δ9,12,15)). Linolenic acid is formed by theactivity of an ω-3-desaturase (Tocher et al. 1998, Prog. Lipid Res. 37,73-117; Domergue et al. 2002, Eur. J. Biochem. 269, 4105-4113).

Mammals, and thus also humans, have no corresponding desaturase activity(Δ12- and ω-3-desaturase) and must take up these fatty acids (essentialfatty acids) via the food. Starting with these precursors, thephysiologically important polyunsaturated fatty acids arachidonic acid(=ARA, 20:4^(Δ5,8,11,14)), an ω-6-fatty acid and the two ω-3-fatty acidseicosapentaenoic acid (=EPA, 20:5^(Δ5,8,11,14,17)) and docosahexaenoicacid (DHA, 22:6^(Δ4,7,10,13,17,19)) are synthesized via the sequence ofdesaturase and elongase reactions. The application of ω-3-fatty acidsshows the therapeutic activity described above in the treatment ofcardiovascular diseases (Shimikawa 2001, World Rev. Nutr. Diet. 88,100-108), inflammations (Calder 2002, Proc. Nutr. Soc. 61, 345-358) andarthritis (Cleland and James 2000, J. Rheumatol. 27, 2305-2307).

From the angle of nutritional physiology, it is therefore important toachieve a shift between the ω-6-synthetic pathway and the ω-3-syntheticpathway (see FIG. 1) in the synthesis of polyunsaturated fatty acids sothat more ω-3-fatty acids are produced. The enzymatic activities ofvarious ω-3-desaturases which desaturate C_(18:2)-, C_(22:4)- orC_(22:5)-fatty acids have been described in the literature (see FIG. 1).However, none of the desaturases whose biochemistry has been describedconverts a broad range of substrates of the ω-6-synthetic pathway intothe corresponding fatty acids of the ω-3-synthetic pathway.

There is therefore still a great demand for an ω-3-desaturase which issuitable for the production of ω-3-polyunsaturated fatty acids. All theknown plant and cyanobacterial ω-3-desaturases desaturate C18-fattyacids with linoleic acid as the substrate, but cannot desaturate C20- orC22-fatty acids.

An ω-3-desaturase which can desaturate C20-polyunsaturated fatty acidsis known from the fungus Saprolegnia dicilina (Pereira et al. 2003,Biochem. J. 2003 Dez, manuscript BJ20031319). However, it isdisadvantageous that this ω-3-desaturase cannot desaturate C18- orC22-PUFAs, such as the important fatty acids C18:2-, C22:4- orC22:5-fatty acids of the ω-6-synthetic pathway. A further disadvantageof this enzyme is that it cannot desaturate fatty acids which are boundto phospholipids. Only the CoA-fatty acid esters are converted.

To make possible the fortification of food and/or of feed with thesepolyunsaturated ω-3-fatty acids, there is therefore a great need for asimple, inexpensive process for the production of these polyunsaturatedfatty acids, especially in eukaryotic systems.

BRIEF SUMMARY OF THE INVENTION

The object of the invention was therefore to provide further genes orenzymes which are suitable for the synthesis of LCPUFAs and which allowa shift from the ω-6-synthetic pathway to the ω-3-synthetic pathway,specifically genes which have an ω-3-desaturase activity, for theproduction of polyunsaturated fatty acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows various synthetic pathways for the biosynthesis of DHA(docosahexaenoic acid).

FIG. 2 shows desaturation of linoleic acid (18:2 ω6-fatty acid) toα-linolenic acid (18:3 ω3-fatty acid) by Pi-omega3Des.

FIG. 3 shows desaturation of γ-linolenic acid (18:3 ω6-fatty acid) tostearidonic acid (18:4 ω3-fatty acid) by Pi-omega3Des.

FIG. 4 shows desaturation of C20:2 ω6-fatty acid to C20:3 ω3-fatty acidby Pi-omega3Des.

FIG. 5 shows desaturation of C20:3 ω6-fatty acid to C20:4 ω3-fatty acidby Pi-omega3Des.

FIG. 6 shows desaturation of arachidonic acid (C20:4 ω6-fatty acid) toeicosapentaenoic acid (C20:5 ω3-fatty acid) by Pi-omega3Des.

FIG. 7 shows desaturation of docosatetraenoic acid (C22:4 ω6-fatty acid)to docosapentaenoic acid (C22:5 ω3-fatty acid) by Pi-omega3Des.

FIG. 8 shows substrate specificity of Pi-omega3Des with regard to avariety of fatty acids.

FIG. 9 shows desaturation of phospholipid-bound arachidonic acid to EPAby Pi-Omega3Des.

DETAILED DESCRIPTION OF THE INVENTION

The object of the invention was furthermore to develop a process for theproduction of polyunsaturated ω-3-fatty acids in an organism,advantageously in a eukaryotic organism, preferably a plant or amicroorganism. This object was achieved by the process according to theinvention for the production of compounds of the general formula I

in transgenic organisms with a content of at least 1% by weight of thesecompounds based on the total lipid content of the transgenic organism,which comprises the following process steps:

-   a) introducing, into the organism, at least one nucleic acid    sequence which encodes an ω-3-desaturase activity, and-   b) culturing the organism under conditions which permits the    production of compounds of the general formula I, and    where the variables and substituents in formula I have the following    meanings:-   R¹=hydroxyl, coenzyme A (thioester), lysophosphatidylcholine,    lysophosphatidylethanolamine, lysophosphatidylglycerol,    lysodiphosphatidylglycerol, lysophosphatidylserine,    lysophosphatidylinositol, sphingo base or a radical of the formula    II

-   R²=hydrogen, lysophosphatidylcholine, lysophosphatidylethanolamine,    lysophosphatidylglycerol, lysodiphosphatidylglycerol,    lysophosphatidylserine, lysophosphatidylinositol or saturated or    unsaturated C₂-C₂₄-alkylcarbonyl,-   R³=hydrogen, saturated or unsaturated C₂-C₂₄-alkylcarbonyl, or R²    and R³ independently of one another are a radical of the formula Ia:

n=2, 3, 4, 5, 6, 7 or 9, m=2, 3, 4, 5 or 6 and p=0 or 3.R¹ in the general formula I is hydroxyl, coenzyme A (thioester),lysophosphatidylcholine, lysophosphatidylethanolamine,lysophosphatidylglycerol, lysodiphosphatidylglycerol,lysophosphatidylserine, lysophosphatidylinositol, sphingo base or aradical of the formula II

The abovementioned radicals of R¹ are always bonded to the compounds ofthe general formula I in the form of their thioesters.

R² in the general formula II is hydrogen, lysophosphatidylcholine,lysophosphatidylethanolamine, lysophosphatidylglycerol,lysodiphosphatidylglycerol, lysophosphatidylserine,lysophosphatidylinositol or saturated or unsaturatedC₂-C₂₄-alkylcarbonyl.

Alkyl radicals which may be mentioned are substituted or unsubstituted,saturated or unsaturated C₂-C₂₄-alkylcarbonyl chains such asethylcarbonyl, n-propylcarbonyl, n-butylcarbonyl, n-pentylcarbonyl,n-hexylcarbonyl, n-heptylcarbonyl, n-octylcarbonyl, n-nonylcarbonyl,n-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl,n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl,n-hexadecylcarbonyl, n-heptadecylcarbonyl, n-octadecylcarbonyl-,n-nonadecylcarbonyl, n-eicosylcarbonyl, n-docosanylcarbonyl- orn-tetracosanylcarbonyl, which comprise one or more double bonds.Saturated or unsaturated C₁₀-C₂₂-alkylcarbonyl radicals such asn-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl,n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl,n-hexadecylcarbonyl, n-heptadecylcarbonyl, n-octadecylcarbonyl,n-nonadecylcarbonyl, n-eicosylcarbonyl, n-docosanylcarbonyl orn-tetracosanylcarbonyl, which comprise one or more double bonds, arepreferred. Especially preferred are saturated and/or unsaturatedC₁₀-C₂₂-alkylcarbonyl radicals such as C₁₀-alkylcarbonyl,C₁₁-alkylcarbonyl, C₁₂-alkylcarbonyl, C₁₃-alkylcarbonyl,C₁₄-alkylcarbonyl, C₁₆-alkylcarbonyl, C₁₈-alkylcarbonyl,C₂₀-alkylcarbonyl or C₂₂-alkylcarbonyl radicals which comprise one ormore double bonds. Very especially preferred are saturated orunsaturated C₁₆-C₂₂-alkylcarbonyl radicals such as C₁₆-alkylcarbonyl,C₁₈-alkylcarbonyl, C₂₀-alkylcarbonyl or C₂₂-alkylcarbonyl radicals whichcomprise one or more double bonds. These advantageous radicals cancomprise two, three, four, five or six double bonds. The especiallyadvantageous radicals with 18, 20 or 22 carbon atoms in the fatty acidchain comprise up to six double bonds, advantageously two, three, fouror five double bonds, especially preferably two, three or four doublebonds. All the abovementioned radicals are derived from thecorresponding fatty acids.

R³ in the formula II is hydrogen, saturated or unsaturatedC₂-C₂₄-alkylcarbonyl.

Alkyl radicals which may be mentioned are substituted or unsubstituted,saturated or unsaturated C₂-C₂₄-alkylcarbonyl chains such asethylcarbonyl, n-propylcarbonyl, n-butylcarbonyl-, n-pentylcarbonyl,n-hexyl carbonyl, n-heptylcarbonyl, n-octylcarbonyl, n-nonylcarbonyl,n-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl,n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl,n-hexadecylcarbonyl, n-heptadecylcarbonyl, n-octadecylcarbonyl-,n-nonadecylcarbonyl, n-eicosylcarbonyl, n-docosanylcarbonyl- orn-tetracosanylcarbonyl, which comprise one or more double bonds.Saturated or unsaturated C₁₀-C₂₂-alkylcarbonyl radicals such asn-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl,n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl,n-hexadecylcarbonyl, n-heptadecylcarbonyl, n-octadecylcarbonyl,n-nonadecylcarbonyl, n-eicosylcarbonyl, n-docosanylcarbonyl orn-tetracosanylcarbonyl, which comprise one or more double bonds, arepreferred. Especially preferred are saturated and/or unsaturatedC₁₀-C₂₂-alkylcarbonyl radicals such as C₁₀-alkylcarbonyl,C₁₁-alkylcarbonyl, C₁₂-alkylcarbonyl, C₁₃-alkylcarbonyl,C₁₄-alkylcarbonyl, C₁₆-alkylcarbonyl, C₁₈-alkylcarbonyl,C₂₀-alkylcarbonyl or C₂₂-alkylcarbonyl radicals which comprise one ormore double bonds. Very especially preferred are saturated orunsaturated C₁₆-C₂₂-alkylcarbonyl radicals such as C₁₆-alkylcarbonyl,C₁₈-alkylcarbonyl, C₂₀-alkylcarbonyl or C₂₂-alkylcarbonyl radicals whichcomprise one or more double bonds. These advantageous radicals cancomprise two, three, four, five or six double bonds. The especiallyadvantageous radicals with 18, 20 or 22 carbon atoms in the fatty acidchain comprise up to six double bonds, advantageously two, three, fouror five double bonds, especially preferably two, three or four doublebonds. All the abovementioned radicals are derived from thecorresponding fatty acids.

The abovementioned radicals of R¹, R² and R³ can be substituted byhydroxyl and/or epoxy groups and/or can comprise triple bonds.

The polyunsaturated fatty acids produced in the process according to theinvention advantageously comprise at least two, advantageously three,four, five or six, double bonds. The fatty acids especiallyadvantageously comprise two, three, four or five double bonds. Fattyacids produced in the process advantageously have 18, 20 or 22 C atomsin the fatty acid chain; the fatty acids preferably comprise 20 or 22carbon atoms in the fatty acid chain. Saturated fatty acids areadvantageously reacted to a minor degree, or not at all, by the nucleicacids used in the process. To a minor degree is to be understood asmeaning that the saturated fatty acids are reacted with less than 5% ofthe activity, advantageously less than 3%, especially advantageouslywith less than 2% of the activity in comparison with polyunsaturatedfatty acids. These fatty acids which have been produced can be producedin the process as a single product or be present in a fatty acidmixture.

The nucleic acid sequences used in the process according to theinvention take the form of isolated nucleic acid sequences which encodepolypeptides with ω-3-desaturase activity.

Nucleic acid sequences which are advantageously used in the processaccording to the invention are nucleic acid sequences which encodepolypeptides with ω-3-desaturase activity selected from the groupconsisting of:

-   a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 1,    or-   b) nucleic acid sequences which, as the result of the degeneracy of    the genetic code, can be derived from the amino acid sequences shown    in SEQ ID NO: 2, or-   c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 1,    which encode polypeptides with at least 60% identity at the amino    acid level with SEQ ID NO: 2, and which have an ω-3-desaturase    activity.

Advantageously, the substituents R² or R³ in the general formulae I andII independently of one another are saturated or unsaturatedC₁₈-C₂₂-alkylcarbonyl; especially advantageously, are independently ofone another unsaturated C₁₈-, C₂₀- or C₂₂-alkylcarbonyl with at leasttwo double bonds.

The polyunsaturated fatty acids produced in the process areadvantageously bound in membrane lipids and/or triacylglycerides, butmay also occur in the organisms as free fatty acids or else bound in theform of other fatty acid esters. In this context, they may be present as“pure products” or else advantageously in the form of mixtures ofvarious fatty acids or mixtures of different glycerides. The variousfatty acids which are bound in the triacylglycerides can be derived fromshort-chain fatty acids with 4 to 6 C atoms, medium-chain fatty acidswith 8 to 12 C atoms or long-chain fatty acids with 14 to 24 C atoms;preferred are the long-chain fatty acids, especially preferred are thelong-chain fatty acids LCPUFAs of C₁₈-, C₂₀- and/or C₂₂-fatty acids.

The process according to the invention advantageously yields fatty acidesters with polyunsaturated C₁₈-, C₂₀- and/or C₂₂-fatty acid moleculeswith at least two double bonds in the fatty acid ester, advantageouslywith at least two, three, four, five or six double bonds in the fattyacid ester, especially advantageously of at least three, four, five orsix double bonds in the fatty acid ester, advantageously leading to thesynthesis of linolenic acid (=LA, C18:2^(Δ9,12,15)), γ-linolenic acid(=GLA, C18:3^(Δ6,9,12)), stearidonic acid (=SDA, C18:4^(Δ6,9,12,15)),dihomo-γ-linolenic acid (=DGLA, 20:3^(Δ8,11,14)) ω-3-eicosatetraenoicacid (=ETA, C20:4^(Δ8,11,14,17)), arachidonic acid (ARA,C20:4^(Δ5,8,11,14)), eicosapentaenoic acid (EPA, C20:5^(Δ5,8,11,14,17)),ω-6-docosapentaenoic acid (C22:5^(Δ4,7,10,13,16)), ω-3-docosapentaenoicacid (C22:5^(Δ4,7,10,13,16,19))docosahexaenoic acid (=DHA,C22:6^(Δ4,7,10,13,16,19)), or their mixtures, preferably ARA, EPA and/orDHA.

The fatty acid esters with polyunsaturated C₁₈-, C₂₀- and/or C₂₂-fattyacid molecules can be isolated in the form of an oil or lipid, forexample in the form of compounds such as sphingolipids,phosphoglycerides, lipids, glycolipids such as glycosphingolipids,phospholipids such as phosphatidylethanolamine, phosphatidylcholine,phosphatidylserine, phosphatidylglycerol, phosphatidylinositol ordiphosphatidylglycerol, monoacylglycerides, diacylglycerides,triacylglycerides or other fatty acid esters such as the acetyl-coenzymeA esters which comprise the polyunsaturated fatty acids with at leasttwo, three, four, five or six, preferably five or six, double bonds,from the organisms which were used for the preparation of the fatty acidesters; preferably, they are isolated in the form of theirdiacylglycerides, triacylglycerides and/or in the form ofphosphatidylcholine, especially preferably in the form of thetriacylglycerides. In addition to these esters, the polyunsaturatedfatty acids are also present in the organisms, preferably in the plants,as free fatty acids or bound in other compounds. As a rule, the variousabovementioned compounds (fatty acid esters and free fatty acids) arepresent in the organisms with an approximate distribution of 80 to 90%by weight of triglycerides, 2 to 5% by weight of diglycerides, 5 to 10%by weight of monoglycerides, 1 to 5% by weight of free fatty acids, 2 to8% by weight of phospholipids, the total of the various compoundsamounting to 100% by weight.

In the process according to the invention, the LCPUFAs which have beenproduced are produced in a content of at least 3% by weight,advantageously at least 5% by weight, preferably at least 8% by weight,especially preferably at least 10% by weight, very especially preferablyat least 15% by weight, based on the total fatty acids in the transgenicorganisms, advantageously in a transgenic plant. The fatty acids areadvantageously produced in bound form. It is possible, with the aid ofthe nucleic acids used in the process according to the invention, forthese unsaturated fatty acids to be positioned at the sn1, sn2 and/orsn3 position of the triglycerides which have advantageously beenproduced. In the PUFA production process, the ω-3-desaturase sequencesaccording to the invention are advantageously used in combination withfurther genes of the PUFA synthesis, such as the Δ4-desaturase,Δ5-desaturase, Δ6-desaturase, Δ8-desaturase, Δ5-elongase, Δ6-elongaseand/or Δ9-elongase gene. This is how, in the process according to theinvention, the end products of the process, for example arachidonic acid(ARA), eicosapentaenoic acid (EPA), ω-6-docosapentaenoic acid or DHA areproduced from the starting compounds linoleic acid (C18:2) or linolenicacid (C18:3) via a number of reaction steps. As a rule, these are notgenerated as absolutely pure products, small traces of the precursorsbeing, as a rule, also present in the end product. If, for example, bothlinoleic acid and linolenic acid are present in the starting organism,or the starting plant, the end products, such as ARA, EPA or DHA, arepresent as mixtures. The precursors should advantageously not amount tomore than 20% by weight, preferably not to more than 15% by weight,especially preferably not to more than 10% by weight, very especiallypreferably not to more than 5% by weight, based on the amount of the endproduct in question. Advantageously, only ARA, EPA or only DHA, bound oras free acids, are produced as end products in the process of theinvention in a transgenic plant. If the compounds ARA, EPA and DHA areproduced simultaneously, they are advantageously produced in a ratio ofat least 1:1:2 (EPA:ARA:DHA), advantageously at least 1:1:3, preferably1:1:4, especially preferably 1:1:5.

Fatty acid esters or fatty acid mixtures produced by the processaccording to the invention advantageously comprise 6 to 15% of palmiticacid, 1 to 6% of stearic acid, 7-85% of oleic acid, 0.5 to 8% ofvaccenic acid, 0.1 to 1% of arachic acid, 7 to 25% of saturated fattyacids, 8 to 85% of monounsaturated fatty acids and 60 to 85% ofpolyunsaturated fatty acids, in each case based on 100% and on the totalfatty acid content of the organisms. Arachidonic acid as advantageouspolyunsaturated fatty acid is present in the fatty acid esters or fattyacid mixtures in a concentration of preferably at least 0.1; 0.2; 0.3;0.4; 0.5; 0.6; 0.7; 0.8; 0.9 or 1%, based on the total fatty acidcontent. Moreover, the fatty acid esters or fatty acid mixtures whichhave been produced by the process of the invention advantageouslycomprise fatty acids selected from the group of the fatty acids erucicacid (13-docosaenoic acid), sterculic acid(9,10-methyleneoctadec-9-enoic acid), malvalic acid(8,9-methyleneheptadec-8-enoic acid), chaulmoogric acid(cyclopentenedodecanoic acid), furan fatty acid(9,12-epoxyoctadeca-9,11-dienoic acid), vernolic acid(9,10-epoxyoctadec-12-enoic acid), tariric acid (6-octadecynoic acid),6-nonadecynoic acid, santalbic acid (t11-octadecen-9-ynoic acid),6,9-octadecenynoic acid, pyrulic acid (t10-heptadecen-8-ynoic acid),crepenyninic acid (9-octadecen-12-ynoic acid), 13,14-dihydrooropheicacid, octadecen-13-ene-9,11-diynoic acid, petroselenic acid(cis-6-octadecenoic acid), 9c,12t-octadecadienoic acid, calendulic acid(8t10t12c-octadecatrienoic acid), catalpic acid(9t11t13c-octadecatrienoic acid), eleostearic acid(9c11t13t-octadecatrienoic acid), jacaric acid(8c10t12c-octadecatrienoic acid), punicic acid(9c11t13c-octadecatrienoic acid), parinaric acid(9c11t13t15c-octadecatetraenoic acid), pinolenic acid(all-cis-5,9,12-octadecatrienoic acid), laballenic acid(5,6-octadecadienallenic acid), ricinoleic acid (12-hydroxyoleic acid)and/or coriolic acid (13-hydroxy-9c,11t-octadecadienoic acid). The fattyacid esters or fatty acid mixtures produced by the process according tothe invention advantageously comprise less than 0.1%, based on the totalfatty adds, or no butyric acid, no cholesterol, no clupanodonic acid(=docosapentaenoic acid, C22:5^(Δ4,8,12,15,21)) and no nisinic acid(tetracosahexaenoic acid, C23:6^(Δ3,8,12,15,18,21)).

Owing to the nucleic acid sequences according to the invention ornucleic acid sequences used in the process according to the invention,an increase in the yield of polyunsaturated ω-3-fatty acids of at least50%, advantageously at least 80%, especially advantageously at least100%, very especially advantageously at least 150% in comparison withthe nontransgenic starting organism, for example a yeast, an alga, afungus or a plant such as Arabidopsis or flax when compared by means ofGC analysis; see Examples.

Chemically pure polyunsaturated fatty acids or fatty acid compositionscan also be synthesized by the processes described above. To this end,the fatty acids or the fatty acid compositions are isolated from theorganism such as the microorganisms or the plants or the culture mediumin which or on which the organisms have been cultured, or from theorganism and the culture medium in the known manner, for example viaextraction, distillation, crystallization, chromatography or acombination of these methods. These chemically pure fatty acids or fattyacid compositions are advantageous for applications in the food industrysector, the cosmetic sector and especially the pharmacological industrysector.

A suitable organism for the production in the process according to theinvention is, in principle, any organism such as microorganisms,nonhuman animals or plants.

Plants which are suitable are, in principle, all those plants which arecapable of synthesizing fatty acids, such as all dicotyledonous ormonocotyledonous plants, algae or mosses. Advantageous plants areselected from the group of the plant families Adelotheciaceae,Anacardiaceae, Asteraceae, Apiaceae, Betulaceae, Boraginaceae,Brassicaceae, Bromeliaceae, Caricaceae, Cannabaceae, Convolvulaceae,Chenopodiaceae, Crypthecodiniaceae, Cucurbitaceae, Ditrichaceae,Elaeagnaceae, Ericaceae, Euphorbiaceae, Fabaceae, Geraniaceae,Gramineae, Juglandaceae, Lauraceae, Leguminosae, Linaceae,Prasinophyceae or vegetable plants or ornamentals such as Tagetes.

Examples which may be mentioned are the following plants selected fromthe group consisting of: Adelotheciaceae such as the generaPhyscomitrella, such as the genus and species Physcomitrella patens,Anacardiaceae such as the genera Pistacia, Mangifera, Anacardium, forexample the genus and species Pistacia vera [pistachio], Mangifer indica[mango] or Anacardium occidentale [cashew], Asteraceae, such as thegenera Calendula, Carthamus, Centaurea, Cichorium, Cynara, Helianthus,Lactuca, Locusta, Tagetes, Valeriana, for example the genus and speciesCalendula officinalis [common marigold], Carthamus tinctorius[safflower], Centaurea cyanus [cornflower], Cichorium intybus [chicory],Cynara scolymus [artichoke], Helianthus annus [sunflower], Lactucasativa, Lactuca crispa, Lactuca esculenta, Lactuca scariola L. ssp.sativa, Lactuca scariola L. var. integrata, Lactuca scariola L. var,integrifolia, Lactuca sativa subsp. romana, Locusta communis, Valerianalocusta [salad vegetables], Tagetes lucida, Tagetes erecta or Tagetestenuifolia [african or french marigold], Apiaceae, such as the genusDaucus, for example the genus and species Daucus carota [carrot],Betulaceae, such as the genus Corylus, for example the genera andspecies Corylus avellana or Corylus columa [hazelnut], Boraginaceae,such as the genus Borago, for example the genus and species Boragoofficinalis [borage], Brassicaceae, such as the genera Brassica,Melanosinapis, Sinapis, Arabadopsis, for example the genera and speciesBrassica napus, Brassica rapa ssp. [oilseed rape], Sinapis arvensisBrassica juncea, Brassica juncea var, juncea, Brassica juncea var.crispifolia, Brassica juncea var. foliosa, Brassica nigra, Brassicasinapioides, Melanosinapis communis [mustard], Brassica oleracea [fodderbeet] or Arabidopsis thaliana, Bromeliaceae, such as the genera Anana,Bromelia (pineapple), for example the genera and species Anana comosus,Ananas ananas or Bromelia comosa [pineapple], Caricaceae, such as thegenus Carica, such as the genus and species Carica papaya [pawpaw],Cannabaceae, such as the genus Cannabis, such as the genus and speciesCannabis sativa [hemp], Convolvulaceae, such as the genera Ipomea,Convolvulus, for example the genera and species Ipomoea batatus, Ipomoeapandurata, Convolvulus batatas, Convolvulus tiliaceus, Ipomoeafastigiata, Ipomoea tiliacea, Ipomoea triloba or Convolvulus panduratus[sweet potato, batate], Chenopodiaceae, such as the genus Beta, such asthe genera and species Beta vulgaris, Beta vulgaris var. altissima, Betavulgaris var. Vulgaris, Beta maritima, Beta vulgaris var. perennis, Betavulgaris var. conditiva or Beta vulgaris var. esculenta [sugarbeet],Crypthecodiniaceae, such as the genus Crypthecodinium, for example thegenus and species Cryptecodinium cohnii, Cucurbitaceae, such as thegenus Cucurbita, for example the genera and species Cucurbita maxima,Cucurbita mixta, Cucurbita pepo or Cucurbita moschata [pumpkin/squash],Cymbellaceae such as the genera Amphora, Cymbella, Okedenia,Phaeodactylum, Reimeria, for example the genus and species Phaeodactylumtricornutum, Ditrichaceae such as the genera Ditrichaceae, Astomiopsis,Ceratodon, Chrysoblastella, Ditrichum, Distichium, Eccremidium,Lophidion, Philibertiella, Pleuridium, Saelania, Trichodon,Skottsbergia, for example the genera and species Ceratodon antarcticus,Ceratodon columbiae, Ceratodon heterophyllus, Ceratodon purpureus,Ceratodon purpureus, Ceratodon purpureus ssp. convolutus, Ceratodon,purpureus spp. stenocarpus, Ceratodon purpureus var. rotundifolius,Ceratodon ratodon, Ceratodon stenocarpus, Chrysoblastella chilensis,Ditrichum ambiguum, Ditrichum brevisetum, Ditrichum crispatissimum,Ditrichum difficile, Ditrichum falcifolium, Ditrichum flexicaule,Ditrichum giganteum, Ditrichum heteromallum, Ditrichum lineare,Ditrichum lineare, Ditrichum montanum, Ditrichum montanum, Ditrichumpallidum, Ditrichum punctulatum, Ditrichum pusillum, Ditrichum pusillumvar. tortile, Ditrichum rhynchostegium, Ditrichum schimperi, Ditrichumtortile, Distichium capillaceum, Distichium hagenii, Distichiuminclinatum, Distichium macounii, Eccremidium flotidanum, Eccremidiumwhiteleggei, Lophidion strictus, Pleuridium acuminatum, Pleuridiumaltemifolium, Pleuridium holdtidgei, Pleuridium mexicanum, Pleuridiumravenelii, Pleuridium subulatum, Saelania glaucescens, Trichodonborealis, Trichodon cylindricus or Trichodon cylindricus var. oblongus,Elaeagnaceae such as the genus Elaeagnus, for example the genus andspecies Olea europaea [olive], Ericaceae such as the genus Kalmia, forexample the genera and species Kalmia latifolia, Kalmia angustifolia,Kalmia microphylla, Kalmia polifolia, Kalmia occidentalis, Cistuschamaerhodendros or Kalmia lucida [mountain laurel], Euphorbiaceae suchas the genera Manihot, Janipha, Jatropha, Ricinus, for example thegenera and species Manihot utilissima, Janipha manihot, Jatrophamanihot, Manihot aipil, Manihot dulcis, Manihot manihot, Manihotmelanobasis, Manihot esculenta [manihot] or Ricinus communis [castor-oilplant], Fabaceae such as the genera Pisum, Albizia, Cathormion,Feuillea, Inga, Pithecolobium, Acacia, Mimosa, Medicajo, Glycine,Dolichos, Phaseolus, Soja, for example the genera and species Pisumsativum, Pisum arvense, Pisum humile [pea], Albizia berteriana, Albiziajulibrissin, Albizia lebbeck, Acacia berteriana, Acacia littoralis,Albizia berteriana, Albizzia berteriana, Cathotmion berteriana, Feuilleaberteriana, Inga fragrans, Pithecellobium berterianum, Pithecellobiumfragrans, Pithecolobium berterianum, Pseudalbizzia berteriana, Acaciajulibrissin, Acacia nemu, Albizia nemu, Feuilleea julibrissin, Mimosajulibrissin, Mimosa speciosa, Sericanrda julibrissin, Acacia lebbeck,Acacia macrophylla, Albizia lebbek, Feuilleea lebbeck, Mimosa lebbeck,Mimosa speciosa [silk tree], Medicago sativa, Medicago falcata, Medicagovaria [alfalfa], Glycine max Dolichos soja, Glycine gracilis, Glycinehispida, Phaseolus max, Soja hispida or Soja max [soybean], Funariaceaesuch as the genera Aphanorrhegma, Entosthodon, Funaria, Physcomitrella,Physcomitrium, for example the genera and species Aphanorrhegmaserratum, Entosthodon attenuatus, Entosthodon bolanderi, Entosthodonbonplandii, Entosthodon californicus, Entosthodon drummondii,Entosthodon jamesonii, Entosthodon leibergii, Entosthodon neoscoticus,Entosthodon rubrisetus, Entosthodon spathulifolius, Entosthodon tucsoni,Funaria americana, Funaria bolanderi, Funaria calcarea, Funariacalifornica, Funaria calvescens, Funaria convoluta, Funaria flavicans,Funaria groutiana, Funaria hygrometrica, Funaria hygrometrica var.arctica, Funaria hygrometrica var. calvescens, Funaria hygrometrica var.convoluta, Funaria hygrometrica var. muralis, Funaria hygrometrica var,utahensis, Funaria microstoma, Funaria microstoma var. obtusifolia,Funaria muhlenbergii, Funaria orcuttii, Funaria plano-convexa, Funariapolaris, Funaria ravenelii, Funaria rubriseta, Funaria serrata, Funariasonorae, Funaria sublimbatus, Funaria tucsoni, Physcomitrellacalifornica, Physcomitrella patens, Physcomitrella readeri,Physcomitrium australe, Physcomitrium californicum, Physcomitriumcollenchymatum, Physcomitrium coloradense, Physcomitrium cupuliferum,Physcomitrium drummondii, Physcomitrium eurystomum, Physcomitriumflexifolium, Physcomitrium hookeri, Physcomitrium hookeri var. serratum,Physcomitrium immersum, Physcomitrium kellermanii, Physcomitriummegalocarpum, Physcomitrium pyriforme, Physcomitrium pyriforme var.serratum, Physcomitrium rufipes, Physcomitrium sandbergii, Physcomitriumsubsphaericum, Physcomitrium washingtoniense, Geraniaceae, such as thegenera Pelargonium, Cocos, Oleum, for example the genera and speciesCocos nucifera, Pelargonium grossularioides or Oleum cocois [coconut],Gramineae, such as the genus Saccharum, for example the genus andspecies Saccharum officinarum, Juglandaceae, such as the genera Juglans,Wallia, for example the genera and species Juglans regia, Juglansailanthifolia, Juglans sieboldiana, Juglans cinerea, Wallia cinerea,Juglans bixbyi, Juglans californica, Juglans Juglans intermedia,Juglansjamaicensis, Juglans major, Juglans microcarpa, Juglans nigra orWallia nigra [walnut], Lauraceae, such as the genera Persea, Laurus, forexample the genera and species Laurus nobilis [bay], Persea americana,Persea gratissima or Persea persea [avocado], Leguminosae, such as thegenus Arachis, for example the genus and species Arachis hypogaea[peanut], Linaceae, such as the genera Linum, Adenolinum, for examplethe genera and species Linum usitatissimum, Linum humile, Linumaustriacum, Linum bienne, Linum angustifolium, Linum catharticum, Linumflavum, Linum grandiflorum, Adenolinum grandiflorum, Linum lewisii,Linum narbonense, Linum perenne, Linum perenne var. lewisii, Linumpratense or Linum trigynum [linseed], Lythrarieae, such as the genusPunica, for example the genus and species Punica granatum [pomegranate],Malvaceae, such as the genus Gossypium, for example the genera andspecies Gossypium hirsutum, Gossypium arboreum, Gossypium barbadense,Gossypium herbaceum or Gossypium thurberi [cotton], Marchantiaceae, suchas the genus Marchantia, for example the genera and species Marchantiaberteroana, Marchantia foliacea, Marchantia macropora, Musaceae, such asthe genus Musa, for example the genera and species Musa nana, Musaacuminata, Musa paradisiaca, Musa spp. [banana], Onagraceae, such as thegenera Camissonia, Oenothera, for example the genera and speciesOenothera biennis or Camissonia brevipes [evening primrose], Palmae,such as the genus Elacis, for example the genus and species Elaeisguineensis [oil palm], Papaveraceae, such as the genus Papaver, forexample the genera and species Papaver orientale, Papaver rhoeas,Papaver dubium [poppy], Pedaliaceae, such as the genus Sesamum, forexample the genus and species Sesamum indicum [sesame], Piperaceae, suchas the genera Piper, Artanthe, Peperomia, Steffensia, for example thegenera and species Piper aduncum, Piper amalago, Piper angustifolium,Piper auritum, Piper betel, Piper cubeba, Piper longum, Piper nigrum,Piper retrofractum, Artanthe adunca, Artanthe elongata, Peperomiaelongata, Piper elongatum, Steffensia elongata [cayenne pepper],Poaceae, such as the genera Hordeum, Secale, Avena, Sorghum, Andropogon,Holcus, Panicum, Oryza, Zea (maize), Triticum, for example the generaand species Hordeum vulgare, Hordeum jubatum, Hordeum murinum, Hordeumsecalinum, Hordeum distichon, Hordeum aegiceras, Hordeum hexastichon,Hordeum hexastichum, Hordeum irregulare, Hordeum sativum, Hordeumsecalinum [barley], Secale cereale [rye], Avena sativa, Avena fatua,Avena byzantina, Avena fatua var. sativa, Avena hybrida [oats], Sorghumbicolor, Sorghum halepense, Sorghum saccharatum, Sorghum vulgare,Andropogon drummondii, Holcus bicolor, Holcus sorghum, Sorghumaethiopicum, Sorghum arundinaceum, Sorghum caffrorum, Sorghum cemuum,Sorghum dochna, Sorghum dnimmondii, Sorghum durra, Sorghum guineense,Sorghum lanceolatum, Sorghum nervosum, Sorghum saccharatum, Sorghumsubglabrescens, Sorghum verticilliflorum, Sorghum vulgare, Holcushalepensis, Sorghum miliaceum, Panicum militaceum [millet], Oryzasativa, Oryza latifolia [rice], Zea mays [maize], Triticum aestivum,Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha,Triticum sativum or Triticum vulgare [wheat], Porphyridiaceae, such asthe genera Chroothece, Flintiella, Petrovanella, Porphyridium, Rhodella,Rhodosorus, Vanhoeffenia, for example the genus and species Porphyridiumcruentum, Proteaceae, such as the genus Macadamia, for example the genusand species Macadamia intergrifolia [macadamia], Prasinophyceae such asthe genera Nephroselmis, Prasinococcus, Scherffelia, Tetraselmis,Mantoniella, Ostreococcus, for example the genera and speciesNephroselmis olivacea, Prasinococcus capsulatus, Scherffelia dubia,Tetraselmis chui, Tetraselmis suecica, Mantoniella squamata,Ostreococcus tauri, Rubiaceae such as the genus Cofea, for example thegenera and species Cofea spp., Coffea arabica, Coffea canephora orCoffea liberica [coffee], Scrophulariaceae such as the genus Verbascum,for example the genera and species Verbascum blattaria, VerbascumVerbascum densiflorum, Verbascum lagurus, Verbascum longifolium,Verbascum lychnitis, Verbascum nigrum, Verbascum olympicum, Verbascumphlomoides, Verbascum phoenicum, Verbascum pulverulentum or Verbascumthapsus [mullein], Solanaceae such as the genera Capsicum, Nicotiana,Solanum, Lycopersicon, for example the genera and species Capsicumannuum, Capsicum annuum var. glabriusculum, Capsicum frutescens[pepper], Capsicum annuum [paprika], Nicotiana tabacum, Nicotiana alata,Nicotiana attenuata, Nicotiana glauca, Nicotiana langsdorffii, Nicotianaobtusifolia, Nicotiana quadrivalvis, Nicotiana repanda, Nicotianarustica, Nicotiana sylvestris [tobacco], Solanum tuberosum [potato],Solanum melongena [eggplant], Lycopersicon esculentum, Lycopersiconlycopersicum, Lycopersicon pyriforme, Solanum integrifolium or Solanumlycopersicum [tomato], Sterculiaceae, such as the genus Theobroma, forexample the genus and species Theobroma cacao [cacao] or Theaceae, suchas the genus Camellia, for example the genus and species Camelliasinensis [tea].

Advantageous microorganisms are, for example, fungi selected from thegroup of the families Chaetomiaceae, Choanephoraceae, Cryptococcaceae,Cunninghamellaceae, Demetiaceae, Moniliaceae, Mortierellaceae,Mucoraceae, Pythiaceae, Sacharomycetaceae, Saprolegniaceae,Schizosacharomycetaceae, Sodariaceae or Tuberculariaceae.

Examples which may be mentioned are the following microorganismsselected from the group: Choanephoraceae such as the genera Blakeslea,Choanephora, for example the genera and species Blakeslea trispora,Choanephora cucurbitarum, Choanephora infundibulifera var. cucurbitarum,Mortierellaceae, such as the genus Mortierella, for example the generaand species Mortierella isabellina, Mortierella polycephala, Mortierellaramanniana, Mortierella vinacea, Mortierella zonata, Pythiaceae such asthe genera Phytium, Phytophthora for example the genera and speciesPythium debaryanum, Pythium intermedium, Pythium irregulare, Pythiummegalacanthurn, Pythium paroecandrum, Pythium sylvaticum, Pythiumultimum, Phytophthora cactorum, Phytophthora cinnamomi, Phytophthoracitricola, Phytophthora citrophthora, Phytophthora cryptogea,Phytophthora drechsleri, Phytophthora erythroseptica, Phytophthoralateralis, Phytophthora megasperma, Phytophthora nicotianae,Phytophthora nicotianae var. parasitica, Phytophthora palmivora,Phytophthora parasitica, Phytophthora syringae, Saccharomycetaceae suchas the genera Hansenula, Pichia, Saccharomyces, Saccharomycodes,Yarrowia for example the genera and species Hansenula anomala, Hansenulacalifornica, Hansenula canadensis, Hansenula capsulata, Hansenulaciferril, Hansenula glucozyma, Hansenula henricii, Hansenula holstii,Hansenula minuta, Hansenula nonfermentans, Hansenula philodendri,Hansenula polymorpha, Hansenula satumus, Hansenula subpelliculosa,Hansenula wickerhamii, Hansenula wingei, Pichia alcoholophila, Pichiaangusta, Pichia anomala, Pichia bispora, Pichia burtonii, Pichiacanadensis, Pichia capsulata, Pichia carsonii, Pichia cellobiosa, Pichiaciferrii, Pichia farinosa, Pichia fermentans, Pichia finlandica, Pichiaglucozyma, Pichia guilliermondii, Pichia haplophila, Pichia henricii,Pichia holstii, Pichia jadinii, Pichia lindnerii, Pichiamembranaefaciens, Pichia methanolica, Pichia minuta var. minuta, Pichiaminuta var. nonfemientans, Pichia norvegensis, Pichia ohmeri, Pichiapastoris, Pichia philodendri, Pichia pini, Pichia polymorpha, Pichiaquercuum, Pichia rhodanensis, Pichia sargentensis, Pichia stipitis,Pichia strasburgensis, Pichia subpelliculosa, Pichia toletana, Pichiatrehalophila, Pichia vini, Pichia xylosa, Saccharomyces aceti,Saccharomyces bailii, Saccharomyces bayanus, Saccharomyces bisporus,Saccharomyces capensis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces cerevisiae var. ellipsoideus, Saccharomyceschevalieri, Saccharomyces delbrueckii, Saccharomyces diastaticus,Saccharomyces drosophilarum, Saccharomyces elegans, Saccharomycesellipsoideus, Saccharomyces fermentati, Saccharomyces florentinus,Saccharomyces fragilis, Saccharomyces heterogenicus, Saccharomyceshienipiensis, Saccharomyces inusitatus, Saccharomyces italicus,Saccharomyces kluyveri, Saccharomyces krusei, Saccharomyces lactis,Saccharomyces marxianus, Saccharomyces microellipsoides, Saccharomycesmontanus, Saccharomyces norbensis, Saccharomyces oleaceus, Saccharomycesparadoxus, Saccharomyces pastorianus, Saccharomyces pretoriensis,Saccharomyces rosei, Saccharomyces Saccharomyces uvarum, Saccharomycodesludwigii, Yarrowia lipolytica, Schizosacharomycetaceae such as thegenera Schizosaccharomyces e.g. the species Schizosaccharomycesjaponicus var. japonicus, Schizosaccharomyces japonicus var. versatilis,Schizosaccharomyces malidevorans, Schizosaccharomyces octosporus,Schizosaccharomyces pombe var. malidevorans, Schizosaccharomyces pombevar. pombe, Thraustochytriaceae such as the genera Althornia,Aplanochytrium, Japonochytrium, Schizochytrium, Thraustochytrium e.g.the species Schizochytrium aggregatum, Schizochytrium limacinum,Schizochytrium mangrovei, Schizochytrium minutum, Schizochytriumoctosporum, Thraustochytrium aggregatum, Thraustochytrium amoeboideum,Thraustochytrium antacticum, Thraustochytrium arudimentale,Thraustochytrium aureum, Thraustochytrium benthicola, Thraustochytriumglobosum, Thraustochytrium indicum, Thraustochytrium kerguelense,Thraustochytrium kinnei, Thraustochytrium motivum, Thraustochytriummultirudimentale, Thraustochytrium pachydermum, Thraustochytriumproliferum, Thraustochytrium roseum, Thraustochytrium rossii,Thraustochytrium striatum or Thraustochytrium visurgense.

Further advantageous microorganisms are, for example, bacteria selectedfrom the group of the families Bacillaceae, Enterobacteriacae orRhizobiaceae.

Examples which may be mentioned are the following microorganismsselected from the group: Bacillaceae such as the genera Bacillus forexample the genera and species Bacillus acidocaldarius, Bacillusacidoterrestris, Bacillus alcalophilus, Bacillus amyloliquefaciens,Bacillus amylolyticus, Bacillus brevis, Bacillus cereus, Bacilluscirculans, Bacillus coagulans, Bacillus sphaericus subsp. fusiformis,Bacillus galactophilus, Bacillus globisporus, Bacillus globisporussubsp. marinus, Bacillus halophilus, Bacillus lentimorbus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus polymyxa,Bacillus psychrosaccharolyticus, Bacillus pumilus, Bacillus sphaericus,Bacillus subtilis subsp. spizizenii, Bacillus subtilis subsp. subtilisor Bacillus thuringiensis; Enterobacteriacae such as the generaCitrobacter, Edwardsiella, Enterobacter, Erwinia, Escherichia,Klebsiella, Salmonella or Serratia for example the genera and speciesCitrobacter amalonaticus, Citrobacter diversus, Citrobacter freundii,Citrobacter genomospecies, Citrobactergillenii, Citrobacter intermedium,Citrobacter koseri, Citrobacter murliniae, Citrobacter sp., Edwardsiellahoshinae, Edwardsiella ictaluri, Edwardsiella tarda, Erwinia alni,Erwinia amylovora, Erwinia ananatis, Erwinia aphidicola, Erwiniabillingiae, Erwinia cacticida, Erwinia cancerogena, Erwinia carnegieana,Erwinia carotovora subsp. atroseptica, Erwinia carotovora subsp.betavasculorum, Erwinia carotovora subsp. odorifera, Erwinia carotovorasubsp. wasabiae, Erwinia chrysanthemi, Erwinia cypripedii, Erwiniadissolvens, Erwinia herbicola, Erwinia mallotivora, Erwinia milletiae,Erwinia nigrifluens, Erwinia nimipressuralis, Erwinia persicina, Erwiniapsidii, Erwinia pyrifoliae, Erwinia quercina, Erwinia rhapontici,Erwinia rubrifaciens, Erwinia salicis, Erwinia stewartii, Erwiniatracheiphila, Erwinia uredovora, Escherichia adecarboxylata, Escherichiaanindolica, Escherichia aurescens, Escherichia blattae, Escherichiacoli, Escherichia coli var. communior, Escherichia coli-mutabile,Escherichia fergusonii, Escherichia hermannii, Escherichia sp.,Escherichia vulneris, Klebsiella aerogenes, Klebsiella edwardsii subsp.atlantae, Klebsiella omithinolytica, Klebsiella oxytoca, Klebsiellaplanticola, Klebsiella pneumoniae, Klebsiella pneumoniae subsp.pneumoniae, Klebsiella sp., Klebsiella terrigena, Klebsiella trevisanii,Salmonella abony, Salmonella arizonae, Salmonella bongori, Salmonellacholeraesuis subsp. arizonae, Salmonella choleraesuis subsp. bongori,Salmonella choleraesuis subsp. cholereasuis, Salmonella choleraesuissubsp. diarizonae, Salmonella choleraesuis subsp. houtenae, Salmonellacholeraesuis subsp. indica, Salmonella choleraesuis subsp. salamae,Salmonella daressalaam, Salmonella enterica subsp. houtenae, Salmonellaenterica subsp. salamae, Salmonella enteritidis, Salmonella gallinarum,Salmonella heidelberg, Salmonella panama, Salmonella senftenberg,Salmonella typhimurium, Serratia entomophila, Serratia ficaria, Serratiafonticola, Serratia grimesii, Serratia liquefaciens, Serratiamarcescens, Serratia marcescens subsp. marcescens, Serratia marinorubra,Serratia odorifera, Serratia plymouthensis, Serratia plymuthica,Serratia proteamaculans, Serratia proteamaculans subsp. quinovora,Serratia quinivorans or Serratia rubidaea; Rhizobiaceae such as thegenera Agrobacterium, Carbophilus, Chelatobacter, Ensifer, Rhizobium,Sinorhizobium for example the genera and species Agrobacteriumatlanticum, Agrobacterium ferrugineum, Agrobacterium gelatinovorum,Agrobacterium lanymoorei, Agrobacterium meteori, Agrobacteriumradiobacter, Agrobacterium rhizogenes, Agrobacterium rubi, Agrobacteriumstellulatum, Agrobacterium tumefaciens, Agrobacterium vitis, Carbophiluscarboxidus, Chelatobacter heintzii, Ensifer adhaerens, Ensifer arboris,Ensifer fredii, Ensifer kostiensis, Ensifer kummerowiae, Ensifermedicae, Ensifer meliloti, Ensifer saheli, Ensifer terangae,Ensiferxinjiangensis, Rhizobium ciceri Rhizobium etli, Rhizobium fredii,Rhizobium galegae, Rhizobium gallicum, Rhizobium giardinii, Rhizobiumhainanense, Rhizobium huakuii, Rhizobium huautlense, Rhizobiumindigoferae, Rhizobium japonicum, Rhizobium leguminosarum, Rhizobiumloessense, Rhizobium loti, Rhizobium lupini, Rhizobium mediterraneum,Rhizobium meliloti, Rhizobium mongolense, Rhizobium phaseoli, Rhizobiumradiobacter, Rhizobium rhizogenes, Rhizobium rubi, Rhizobium sullae,Rhizobium tianshanense, Rhizobium trifolii, Rhizobium tropici, Rhizobiumundicola, Rhizobium vitis, Sinorhizobium adhaerens, Sinorhizobiumarboris, Sinorhizobium fredii, Sinorhizobium kostiense, Sinorhizobiumkummerowiae, Sinorhizobium medicae, Sinorhizobium meliloti,Sinorhizobium morelense, Sinorhizobium saheli or Sinorhizobiumxinjiangense.

Further advantageous microorganisms for the process according to theinvention are, for example, protists or diatoms selected from the groupof the families Dinophyceae, Turaniellidae or Oxytrichidae, such as thegenera and species: Crypthecodinium cohnii, Phaeodactylum tricornutum,Stylonychia mytilus, Stylonychia pustulata, Stylonychia putrina,Stylonychia notophora, Stylonychia sp., Colpidium campylum or Colpidiumsp.

Organisms which are advantageously used in the process according to theinvention are transgenic organisms such as fungi, such as Mortierella orTraustochytrium, yeasts such as Saccharomyces or Schizosaccharomyces,mosses such as Physcomitrella or Ceratodon, nonhuman animals such asCaenorhabditis, algae such as Crypthecodinium or Phaeodactylum or plantssuch as dicotyledonous or monocotyledonous plants. Organisms which areespecially advantageously used in the process according to the inventionare organisms which belong to the oil-producing organisms, that is tosay which are used for the production of oils, such as fungi such asMortierella or Thraustochytrium, algae such as Crypthecodinium,Phaeodactylum or plants, in particular plants, preferably, oil fruitcrops which comprise large amounts of lipid compounds, such as peanut,oilseed rape, canola, sunflower, safflower, poppy, mustard, hemp,castor-oil plant, olive, sesame, Calendula, Punica, evening primrose,mullein, thistle, wild roses, hazelnut, almond, macadamia, avocado, bay,pumpkin/squash, linseed, soybean, pistachios, borage, trees (oil palm,coconut, walnut) or crops such as maize, wheat, rye, oats, triticale,rice, barley, cotton, cassaya, pepper, Tagetes, Solanaceae plants suchas potato, tobacco, eggplant and tomato, Vicia species, pea, alfalfa orbushy plants (coffee, cacao, tea), Salix species, and perennial grassesand fodder crops. Preferred plants according to the invention are oilcrop plants such as peanut, oilseed rape, canola, sunflower, safflower,poppy, mustard, hemp, castor-oil plant, olive, Calendula, Punica,evening primrose, pumpkin/squash, linseed, soybean, borage, trees (oilpalm, coconut). Especially preferred are plants which are high in C18:2-and/or C18:3-fatty acids, such as sunflower, safflower, tobacco,mullein, sesame, cotton, pumpkin/squash, poppy, evening primrose,walnut, linseed, hemp, thistle or safflower. Very especially preferredplants are plants such as safflower, sunflower, poppy, evening primrose,walnut, linseed, or hemp.

It is advantageous for the above-described process according to theinvention to introduce, into the organism, further nucleic acids whichencode enzymes of the fatty acid or lipid metabolism, in addition to thenucleic acid sequences introduced in the process which encode anω-3-desaturase.

In principle, all genes of the fatty acid or lipid metabolism can beused in the process for the production of polyunsaturated fatty acids,advantageously in combination with the inventive ω-3-desaturase. Genesof the fatty acid or lipid metabolism selected from the group consistingof acyl-CoA dehydrogenase(s), acyl-ACP [=acyl carrier protein]desaturase(s), acyl-ACP thioesterase(s), fatty acid acyl transferase(s),acyl-CoA:lysophospholipid acyltransferases, fatty acid synthase(s),fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s),acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acidacetylenases, lipoxygenases, triacylglycerol lipases, allenoxidesynthases, hydroperoxide lyases or fatty acid elongase(s) areadvantageously used in combination with the ω-3-desaturase. Genesselected from the group of the Δ4-desaturases, Δ5-desaturases,Δ6-desaturases, Δ8-desaturases, Δ9-desaturases, Δ12-desaturases,Δ5-elongases, Δ6-elongases or Δ9-elongases are especially preferablyused in combination with the above genes for ω-3-desaturase, it beingpossible to use individual genes or a plurality of genes in combination.

In comparison with the known ω-3-desaturase, the ω-3-desaturaseaccording to the invention has the advantageous characteristic that itis capable of desaturating a broad spectrum of ω-6-fatty acids, withC₂₀- and C₂₂-fatty acids such as C_(20:2)-, C_(20:3)-, C_(20:4)-,C_(22:4)- or C_(22:5)-fatty acids being preferentially desaturated.However, the shorter C₁₈-fatty acids such as C_(18:2)- or C_(18:3)-fattyacids are also advantageously desaturated. Owing to thesecharacteristics of ω-3-desaturase, it is advantageously possible toshift the fatty acid spectrum within an organism, advantageously withina plant or a fungus, from the ω-6-fatty acids towards the ω-3-fattyacids. The ω-3-desaturase according to the invention preferentiallydesaturates C₂₀-fatty acids. Within the organism, these fatty acids areconverted to at least 10%, 15%, 20%, 25% or 30% from the existing fattyacid pool to give the corresponding ω-3-fatty acids. In comparison withthe C₁₈-fatty acids, the activity of ω-3-desaturase is lower by a factorof 10, that is to say only approximately 1.5 to 3% of the fatty acidspresent in the fatty acid pool are converted into the correspondingω-3-fatty acids. Preferred substrates of the ω-3-desaturase according tothe invention are the ω-6-fatty acids bound in phospholipids. Withreference to the desaturation of dihomo-γ-linolenic acid [C_(20:4)^(Δ8,11,14)], FIG. 9 shows clearly that ω-3-desaturase advantageouslydoes not differentiate between fatty acids bound at the sn1 or sn2position when desaturation takes place. Both fatty acids bound at thesn1 position and fatty acids bound at the sn2 position in thephospholipids are desaturated. Another advantage is that ω-3-desaturaseconverts a broad range of phospholipids such as phosphatidylcholine(=PC), phosphatidylinositol (=PIS) or phosphatidylethanolamine (=PE).Finally, desaturation products are also found in the neutral lipids(=NL), i.e. in the triglycerides.

Owing to the enzymatic activity of the nucleic acids used in the processaccording to the invention which encode polypeptides with ω-3-desaturaseactivity, advantageously in combination with nucleic acid sequenceswhich encode polypeptides of the fatty acid or lipid metabolism, such asadditionally polypeptides with Δ4-, Δ5-, Δ6-, Δ8-desaturase or Δ5-, Δ6-or Δ9-elongase activity, a very wide range of polyunsaturated fattyacids can be produced in the process according to the invention.Depending on the choice of the organisms, such as the advantageous plantused for the process according to the invention, mixtures of the variouspolyunsaturated fatty acids or individual polyunsaturated fatty acidssuch as EPA or DHA can be produced in free or bound form. Depending onthe prevailing fatty acid composition in the starting plant (C18:2- orC18:3-fatty acids), fatty acids which are derived from C18:2-fattyacids, such as GLA, DGLA or ARA, or which are derived from C18:3-fattyacids, such as SDA, ETA or EPA, are thus obtained. If only linoleic acid(=LA, C18:2^(Δ9,12)) is present as unsaturated fatty acid in the plantused for the process, the process can only initially afford GLA, DGLAand ARA as products, all of which can be present as free fatty acids orin bound form. As a result of the activity of the ω-3-desaturaseaccording to the invention, ω-3-fatty acids can eventually besynthesized from the above. If in the plant used in the process onlyα-linolenic acid (=ALA, C18:3^(Δ9,12,15)) as unsaturated fatty acid, asis the case in flax, the process can only afford SDA, ETA, EPA and/orDHA as products, which, as described above, can be present as free fattyacids or in bound form. Owing to the modification of the activity of theenzyme ω-3-desaturase which plays a role in the synthesis,advantageously in combination with Δ4-, Δ5-, Δ6-desaturase and/orΔ6-elongase, and/or Δ5-elongase, or Δ4-, Δ5-, Δ8-desaturase, and/orΔ9-elongase and/or Δ5-elongase, it is possible to produce, in a targetedfashion, only individual products in the abovementioned organisms,advantageously in the abovementioned plants. Owing to the activity ofΔ6-desaturase and Δ6-elongase, for example, GLA and DGLA, or SDA andETA, are formed, depending on the starting plant and unsaturated fattyacid. DGLA or ETA or mixtures of these are preferentially formed. IfΔ5-desaturase, Δ5-elongase and Δ4-desaturase are additionally introducedinto the organisms, advantageously into the plant, ARA, EPA and/or DHAare additionally formed. This also applies to organisms into whichΔ8-desaturase and Δ9-elongase have previously been introduced.Advantageously, only ARA, EPA or DHA or their mixtures, especiallyadvantageously only EPA and DHA or their mixtures, are synthesized,depending on the fatty acid present in the organism or plant, which actsas starting substance for the synthesis. Since biosynthetic cascades areinvolved, the end products in question are not present in pure form inthe organisms. Small amounts of the precursor compounds are alwaysadditionally present in the end product. These small amounts amount toless than 20% by weight, advantageously less than 15% by weight,especially advantageously less than 10% by weight, very especiallyadvantageously less than 5, 4, 3, 2, or 1% by weight, based on the endproduct DGLA, ETA or their mixtures, or ARA, EPA, DHA or their mixtures,advantageously EPA or DHA or their mixtures.

In addition to the production directly in the organism of the startingfatty acids for the ω-3-desaturase according to the invention, the fattyacids can also be fed externally. The production in the organism ispreferred for reasons of economy. Preferred substrates of ω-3-desaturaseare linoleic acid (C18:2^(Δ9,12)), γ-linolenic acid (C18:3^(Δ6,9,12)),eicosadienoic acid (C20:2^(Δ11,14)), dihomo-γ-linolenic acid(C20:3^(Δ8,11,14)), arachidonic acid (C20:4^(Δ5,8,11,14)),docosatetraenoic acid (C22:4^(Δ7,10,13,16)) and docosapentaenoic acid(C22:5^(Δ4,7,10,13,15)).

To increase the yield in the above-described process for the productionof oils and/or triglycerides with an advantageously elevated content ofpolyunsaturated fatty acids, it is advantageous to increase the amountof starting product for the synthesis of fatty acids; this can beachieved for example by introducing, into the organism, a nucleic acidwhich encodes a polypeptide with Δ12-desaturase activity. This isparticularly advantageous in oil-producing organisms such as oilseedrape which are high in oleic acid. Since these organisms are only low inlinoleic acid (Mikoklajczak et al., Journal of the American Oil ChemicalSociety, 38, 1961, 678-681), the use of the abovementionedΔ12-desaturases for producing the starting material linoleic acid isadvantageous.

Nucleic acids used in the process according to the invention areadvantageously derived from plants such as algae, for exampleIsochrysis, Mantoniella, Ostreococcus or Crypthecodinium, algae/diatomssuch as Phaeodactylum or Thraustochytrium, mosses such as Physcomitrellaor Ceratodon, or higher plants such as the Primulaceae such asAleuritia, Calendula stellata, Osteospermum spinescens or Osteospermumhyoseroides, microorganisms such as fungi, such as Aspergillus,Thraustochytrium, Phytophthora, Entomophthora, Mucor or Mortierella,bacteria such as Shewanella, yeasts or animals such as nematodes such asCaenorhabditis, insects or fish. The isolated nucleic acid sequencesaccording to the invention are advantageously derived from an animal ofthe order of the vertebrates. Preferably, the nucleic acid sequences arederived from the classes of the Vertebrata; Euteleostomi,Actinopterygii; Neopterygii; Teleostei; Euteleostei,Protacanthopterygii, Salmoniformes; Salmonidae or Oncorhynchus. Thenucleic acids are especially advantageously derived from fungi, animals,or from plants such as algae or mosses, preferably from the order of theSalmoniformes, such as the family of the Salmonidae, such as the genusSalmo, for example from the genera and species Oncorhynchus mykiss,Trutta trutta or Salmo trutta fario, or from the diatoms such as thegenera Thallasiosira or Crypthecodinium.

The process according to the invention advantageously employs theabovementioned nucleic acid sequences or their derivatives or homologswhich encode polypeptides which retain the enzymatic activity of theproteins encoded by nucleic acid sequences. These sequences,individually or in combination with the nucleic acid sequences whichencode ω-3-desaturase, are cloned into expression constructs and usedfor the introduction into, and expression in, organisms. Owing to theirconstruction, these expression constructs make possible an advantageousoptimal synthesis of the polyunsaturated fatty acids produced in theprocess according to the invention.

In a preferred embodiment, the process furthermore comprises the step ofobtaining a cell or an intact organism which comprises the nucleic acidsequences used in the process, where the cell and/or the organism istransformed with a nucleic acid sequence according to the inventionwhich encodes the ω-3-desaturase, a gene construct or a vector asdescribed below, alone or in combination with further nucleic acidsequences which encode proteins of the fatty acid or lipid metabolism.In a further preferred embodiment, this process furthermore comprisesthe step of obtaining the oils, lipids or free fatty acids from theorganism or the culture. The culture may, for example, take the form ofa fermentation culture, for example in the case where microorganismssuch as, for example, Mortierella, Saccharomyces or Thraustochytrium arecultured, or the form of a greenhouse or field-grown culture of a plant.The cell thus produced, or the organism thus produced, is advantageouslya cell of an oil-producing organism, such as an oil crop, such as, forexample, peanut, oilseed rap, canola, linseed, hemp, peanut, soybean,safflower, hemp, sunflowers or borage.

In the case of plant cells, plant tissue or plant organs, “growing” isunderstood as meaning, for example, the cultivation on or in a nutrientmedium, or of the intact plant on or in a substrate, for example in ahydroponic culture, potting compost or on arable land.

For the purposes of the invention, “transgenic” or “recombinant” meanswith regard to, for example, a nucleic acid sequence an expressioncassette (=gene construct) or a vector comprising the nucleic acidsequence according to the invention or an organism transformed with thenucleic acid sequences, expression cassette or vector according to theinvention, all those constructions brought about by recombinant methodsin which either

-   a) the nucleic acid sequence according to the invention, or-   b) a genetic control sequence which is operably linked with the    nucleic acid sequence according to the invention, for example a    promoter, or-   c) a) and b)    are not located in their natural genetic environment or have been    modified by recombinant methods, it being possible for the    modification to take the form of, for example, a substitution,    addition, deletion, inversion or insertion of one or more nucleotide    residues. The natural genetic environment is understood as meaning    the natural genomic or chromosomal locus in the original organism or    the presence in a genomic library. In the case of a genomic library,    the natural genetic environment of the nucleic acid sequence is    preferably retained, at least in part. The environment flanks the    nucleic acid sequence at least on one side and has a sequence length    of at least 50 bp, preferably at least 500 bp, especially preferably    at least 1000 bp, most preferably at least 5000 bp. A naturally    occurring expression cassette—for example the naturally occurring    combination of the natural promoter of the nucleic acid sequences    according to the invention with the corresponding ω-3-desaturase    genes—becomes a transgenic expression cassette when this expression    cassette is modified by unnatural, synthetic (“artificial”) methods    such as, for example, mutagenic treatment. Suitable methods are    described, for example, in U.S. Pat. No. 5,565,350 or WO 00/15815.

Transgenic organism or transgenic plant for the purposes of theinvention is, as mentioned above, understood as meaning that the nucleicacids used in the process are not at their natural locus in the genomeof an organism, it being possible for the nucleic acids to be expressedhomologously or heterologously. However, transgenic also means that, asmentioned above, while the nucleic acids according to the invention areat their natural position in the genome of an organism, however, thesequence has been modified with regard to the natural sequence, and/orthat the regulatory sequences of the natural sequences have beenmodified. Transgenic is preferably understood as meaning the expressionof the nucleic acids according to the invention at an unnatural locus inthe genome, i.e. homologous or, preferably, heterologous expression ofthe nucleic acids takes place. Preferred transgenic organisms are fungisuch as Mortierella, mosses such as Physcomitrella, algae such asCrypthecodinium or plants such as the oil crop plants.

Suitable organisms, or host organisms, for the nucleic acids, theexpression cassette or the vector used in the process according to theinvention are advantageously in principle all those organisms which arecapable of synthesizing fatty acids, specifically unsaturated fattyacids, or which are suitable for the expression of recombinant genes.Examples which may be mentioned are plants such as Brassicaceae, such asArabidopsis, Asteraceae such as Calendula or crop plants such assoybean, peanut, castor-oil plant, sunflower, maize, cotton, flax,oilseed rape, coconut, oil palm, safflower (Carthamus tinctorius) orcacao bean, microorganisms such as fungi, for example the genusMortierella, Thraustochytrium, Saprolegnia or Pythium, bacteria such asthe genus Escherichia or Shewanella, yeasts such as the genusSaccharomyces, cyanobacteria, ciliates, algae or protozoans such asdinoflagellates, such as Crypthecodinium. Organisms which are naturallycapable of synthesizing large amounts of oils are preferred, such asfungi such as Mortierella alpina, Pythium insidiosum or plants such assoybean, oilseed rape, coconut, oil palm, safflower, flax, hemp,castor-oil plant, Calendula, peanut, cacao bean or sunflower, or yeastssuch as Saccharomyces cerevisiae, with soybean, flax, oilseed rape,safflower, sunflower, Calendula, Mortierella or Saccharomyces cerevisiaebeing especially preferred. In principle, transgenic animals,advantageously nonhuman animals, for example C. elegans, are alsosuitable as host organisms, in addition to the abovementioned transgenicorganisms.

Host cells which can be exploited are furthermore mentioned in: Goeddel,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990).

Expression strains which can be used, for example those with a lowerprotease activity, are described in: Gottesman, S., Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990) 119-128.

These include plant cells and certain tissues, organs and parts ofplants in all their phenotypic forms such as anthers, fibers, roothairs, stalks, embryos, calli, cotelydons, petioles, harvested material,plant tissue, reproductive tissue and cell cultures which is derivedfrom the actual transgenic plant and/or can be used for bringing aboutthe transgenic plant.

Transgenic plants which comprise the polyunsaturated fatty acidssynthesized in the process according to the invention can advantageouslybe marketed directly without there being any need for the oils, lipidsor fatty acids synthesized to be isolated. Plants for the processaccording to the invention are understood as meaning intact plants andall plant parts, plant organs or plant parts such as leaf, stem, seed,root, tubers, anthers, fibers, root hairs, stalks, embryos, calli,cotelydons, petioles, harvested material, plant tissue, reproductivetissue and cell cultures which are derived from the transgenic plantand/or can be used for bringing about the transgenic plant. In thiscontext, the seed comprises all parts of the seed such as the seedcoats, epidermal cells, seed cells, endosperm or embryonic tissue.However, the compounds produced in the process according to theinvention can also be isolated from the organisms, advantageously theplants, in the form of their oils, fat, lipids and/or free fatty acids.Polyunsaturated fatty acids produced by this process can be harvested byharvesting the organisms either from the culture in which they grow, orfrom the field. This can be done via pressing or extraction of the plantparts, preferably the plant seeds. In this context, the oils, fats,lipids and/or free fatty acids can be obtained by what is known ascold-beating or cold-pressing without applying heat by pressing. Toallow for greater ease of disruption of the plant parts, specificallythe seeds, they are previously comminuted, steamed or roasted. The seedswhich have been pretreated in this manner can subsequently be pressed orextracted with solvent such as warm hexane. The solvent is subsequentlyremoved again. In the case of microorganisms, for example, these areharvested and then extracted directly without further processing steps,or else disrupted and then extracted via various methods with which theskilled worker is familiar. In this manner, more than 96% of thecompounds produced in the process can be isolated. Thereafter, theresulting products are processed further, i.e. refined. In this process,substances such as the plant mucilages and suspended matter are firstremoved. What is known as desliming can be effected enzymatically or,for example, chemico-physically by addition of acid such as phosphoricacid. Thereafter, the free fatty acids are removed by treatment with abase, for example sodium hydroxide solution. The resulting product iswashed thoroughly with water to remove the alkali remaining in theproduct and then dried. To remove the pigment remaining in the product,the products are subjected to bleaching, for example using fuller'searth or active charcoal. At the end, the product is deodorized, forexample using steam.

The PUFAs or LCPUFAs produced by this process are preferably C₁₈-, C₂₀-or C₂₂-fatty acid molecules with at least two double bonds in the fattyacid molecule, preferably with two, three, four, five or six doublebonds. These C₁₈-, C₂₀- or C₂₂-fatty acid molecules can be isolated fromthe organism in the form of an oil, a lipid or a free fatty acid.Examples of suitable organisms are those mentioned above. Preferredorganisms are transgenic plants.

One embodiment of the invention are therefore oils, lipids or fattyacids or fractions thereof which have been prepared by theabove-described process, especially preferably oil, lipid or a fattyacid composition which comprise PUFAs and originate from transgenicplants.

As described above, these oils, lipids or fatty acids advantageouslycomprise 6 to 15% of palmitic acid, 1 to 6% of stearic acid, 7-85% ofoleic acid, 0.5 to 8% of vaccenic acid, 0.1 to 1% of arachic acid, 7 to25% of saturated fatty acids, 8 to 85% of monounsaturated fatty acidsand 60 to 85% of polyunsaturated fatty acids, in each case based on 100%and on the total fatty acid content of the organisms. Advantageouspolyunsaturated fatty acid which is present in the fatty acid esters orfatty acid mixtures is preferably at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9 or 1% of arachidonic acid, based on the total fatty acidcontent. Moreover, the fatty acid esters or fatty acid mixtures whichhave been produced by the process of the invention advantageouslycomprise fatty acids selected from the group of the fatty acids erucicacid (13-docosaenoic acid), sterculic acid(9,10-methyleneoctadec-9-enoic acid), malvalic acid(8,9-methyleneheptadec-8-enoic acid), chaulmoogric acid(cyclopentenedodecanoic acid), furan fatty acid(9,12-epoxyoctadeca-9,11-dienoic acid), vernonic acid(9,10-epoxyoctadec-12-enoic acid), tariric acid (6-octadecynoic acid),6-nonadecynoic acid, santalbic acid (t11-octadecen-9-ynoic acid),6,9-octadecenynoic acid, pyrulic acid (t10-heptadecen-8-ynoic acid),crepenyninic acid (9-octadecen-12-ynoic acid), 13,14-dihydrooropheicacid, octadecen-13-ene-9,11-diynoic acid, petroselenic acid(cis-6-octadecenoic acid), 9c,12t-octadecadienoic acid, calendulic acid(8t10t12c-octadecatrienoic acid), catalpic acid(9t11t13c-octadecatrienoic acid), eleostearic acid(9c11t13t-octadecatrienoic acid), jacaric acid(8c10t12c-octadecatrienoic acid), punicic acid(9c11t13c-octadecatrienoic acid), parinaric acid(9c11t13t15c-octadecatetraenoic acid), pinolenic acid(all-cis-5,9,12-octadecatrienoic acid), laballenic acid(5,6-octadecadienallenic acid), ricinoleic acid (12-hydroxyoleic acid)and/or coriolic acid (13-hydroxy-9c,11t-octadecadienoic acid). The fattyacid esters or fatty acid mixtures produced by the process according tothe invention advantageously comprise less than 0.1%, based on the totalfatty acids, or no butter butyric acid, no cholesterol, no clupanodonicacid (=dpcpsapentaenoic acid, C22:5^(Δ4,8,12,15,21)) and no nisinic acid(tetracosahexaenoic acid, C23:6^(Δ3,8,12,15,18,21)).

The oils, lipids or fatty acids according to the inventionadvantageously comprise at least 0.5%, 1%, 2%, 3%, 4% or 5%,advantageously at least 6%, 7%, 8%, 9% or 10%, especially advantageouslyat least 11%, 12%, 13%, 14% or 15% of ARA or at least 0.5%, 1%, 2%, 3%,4% or 5%, advantageously at least 6% or 7%, especially advantageously atleast 8%, 9% or 10% of EPA and/or of DHA, based on the total fatty acidcontent of the production organism, advantageously of a plant,especially advantageously of an oil crop such as soybean, oilseed rape,coconut, oil palm, safflower, flax, hemp, castor-oil plant, Calendula,peanut, cacao bean, sunflower or the abovementioned othermonocotyledonous or dicotyledonous oil crops.

A further embodiment according to the invention is the use of the oil,lipid, fatty acids and/or the fatty acid composition in feedstuffs,foodstuffs, cosmetics or pharmaceuticals. The oils, lipids, fatty acidsor fatty acid mixtures according to the invention can be used in themanner with which the skilled worker is familiar for mixing with otheroils, lipids, fatty acids or fatty acid mixtures of animal origin suchas, for example, fish oils.

The term “oil”, “lipid” or “fat” is understood as meaning a fatty acidmixture comprising unsaturated or saturated, preferably esterified,fatty acid(s). The oil, lipid or fat is preferably high inpolyunsaturated free or, advantageously, esterified fatty acid(s), inparticular linoleic acid, γ-linolenic acid, dihomo-γ-linolenic acid,arachidonic acid, α-linolenic acid, stearidonic acid, eicosatetraenoicacid, eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoicacid. The amount of unsaturated esterified fatty acids preferablyamounts to approximately 30%, a content of 50% is more preferred, acontent of 60%, 70%, 80% or more is even more preferred. For theanalysis, the fatty acid content can, for example, be determined by gaschromatography after converting the fatty acids into the methyl estersby transesterification. The oil, lipid or fat can comprise various othersaturated or unsaturated fatty acids, for example calendulic acid,palmitic acid, palmitoleic acid, stearic acid, oleic acid and the like.The content of the various fatty acids in the oil or fat can vary, inparticular depending on the starting organism.

The polyunsaturated fatty acids with advantageously at least two doublebonds, which acids are produced in the process, are, as described above,for example sphingolipids, phosphoglycerides, lipids, glycolipids,phospholipids, monoacylglycerol, diacylglycerol, triacylglycerol orother fatty acid esters.

Starting from the polyunsaturated fatty acids with advantageously atleast five or six double bonds, which acids have been prepared in theprocess according to the invention, the polyunsaturated fatty acidswhich are present can be liberated for example via treatment withalkali, for example aqueous KOH or NaOH, or acid hydrolysis,advantageously in the presence of an alcohol such as methanol orethanol, or via enzymatic cleavage, and isolated via, for example, phaseseparation and subsequent acidification via, for example, H₂SO₄. Thefatty acids can also be liberated directly without the above-describedprocessing step.

After their introduction into an organism, advantageously a plant cellor plant, the nucleic acids used in the process can either be present ona separate plasmid or, advantageously, integrated into the genome of thehost cell. In the case of integration into the genome, integration canbe random or else be effected by recombination such that the native geneis replaced by the copy introduced, whereby the production of thedesired compound by the cell is modulated, or by the use of a gene intrans, so that the gene is linked operably with a functional expressionunit which comprises at least one sequence which ensures the expressionof a gene and at least one sequence which ensures the polyadenylation ofa functionally transcribed gene. The nucleic acids are advantageouslyintroduced into the organisms via multiexpression cassettes orconstructs for multiparallel expression, advantageously into the plantsfor the multiparallel seed-specific expression of genes.

Mosses and algae are the only known plant systems which producesubstantial amounts of polyunsaturated fatty acids such as arachidonicacid (ARA) and/or eicosapentaenoic acid (EPA) and/or docosahexaenoicacid (DHA). Mosses comprise PUFAs in membrane lipids, while algae,organisms which are related to algae and a few fungi also accumulatesubstantial amounts of PUFAs in the triacylglycerol fraction. This iswhy nucleic acid molecules which are isolated from such strains whichalso accumulate PUFAs in the triacylglycerol fraction are particularlyadvantageous for the process according to the invention and thus for themodification of the lipid and PUFA production system in a host, inparticular plants such as oil crops, for example oilseed rape, canola,linseed, hemp, soybean, sunflowers and borage. They can therefore beused advantageously in the process according to the invention.

Substrates which are advantageously suitable for the nucleic acids whichare used in the process according to the invention and which encodepolypeptides with ω-3-desaturase activity and/or the further nucleicacids used, such as the nucleic acids which encode polypeptides of thefatty add or lipid metabolism selected from the group acyl-CoAdehydrogenase(s), acyl-ACP [=acyl carrier protein] desaturase(s),acyl-ACP thioesterase(s), fatty acid acyltransferase(s),acyl-CoA:lysophospholipid acyltransferase(s), fatty acid synthase(s),fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s),acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acidacetylenase(s), lipoxygenase(s), triacylglycerol lipase(s), allenoxidesynthase(s), hydroperoxide lyase(s) or fatty acid elongase(s) are C₁₈-,C₂₀- or C₂₂-fatty acids. The fatty acids converted as substrates in theprocess are preferably converted in the form of their phospholipidesters.

To produce the long-chain PUFAs according to the invention, thepolyunsaturated C₁₈-fatty acids must first be desaturated by theenzymatic activity of a desaturase and subsequently be elongated by atleast two carbon atoms via an elongase. After one elongation cycle, thisenzyme activity gives C₂₀-fatty acids and after two elongation cyclesC₂₂-fatty acids. The activity of the desaturases and elongases used inthe process according to the invention preferably leads to C₁₈-, C₂₀-and/or C₂₂-fatty acids, advantageously with at least two double bonds inthe fatty acid molecule, preferably with three, four, five or six doublebonds, especially preferably to give C₂₀- and/or C₂₂-fatty acids with atleast two double bonds in the fatty acid molecule, preferably withthree, four, five or six double bonds, very especially preferably withfour, five or six double bonds in the molecule. After a firstdesaturation and the elongation have taken place, further desaturationand elongation steps, for example such a desaturation in the Δ5 and Δ4positions, may take place. Products of the process according to theinvention which are especially preferred are elcosatrienoic acid,eicosapentaenoic acid, docosapentaenoic acid and/or docosahexaenoicacid. The C₂₀-fatty acids with at least two double bonds in the fattyacid can be elongated by the enzymatic activity of the enzymes used inthe process in the form of the free fatty acid or in the form of theesters, such as phospholipids, glycolipids, sphingolipids,phosphoglycerides, monoacylglycerol, diacylglycerol or triacylglycerol.

The preferred biosynthesis site of fatty acids, oils, lipids or fats inthe plants which are advantageously used is, for example, in general theseed or cell strata of the seed, so that seed-specific expression of thenucleic adds used in the process makes sense. However, it is obviousthat the biosynthesis of fatty acids, oils or lipids need not be limitedto the seed tissue, but can also take place in a tissue-specific mannerin all the other parts of the plant, for example in epidermal cells orin the tubers.

If, in the process according to the invention, microorganisms such asyeasts, such as Saccharomyces or Schizosaccharomyces, fungi such asMortierella, Aspergillus, Phytophthora, Entomophthora, Mucor orThraustochytrium, algae such as Isochrysis, Phaeodactylum orCrypthecodinium are used as organisms, these organisms are preferablycultured in a fermentation.

Owing to the use of the nucleic acids according to the invention whichencode a ω-3-desaturase, the polyunsaturated fatty acids produced in theprocess can be increased by at least 5%, preferably by at least 10%,especially preferably by at least 20%, very especially preferably by atleast 50% in comparison with the wild type of the organisms which do notcomprise the nucleic acids recombinantly.

In principle, the polyunsaturated fatty acids produced by the processaccording to the invention in the organisms used in the process can beincreased in two ways. The pool of free polyunsaturated fatty acidsand/or the content of the esterified polyunsaturated fatty acidsproduced via the process can be advantageously enlarged. Advantageously,the pool of esterified polyunsaturated fatty acids in the transgenicorganisms is enlarged by the process according to the invention.

If microorganisms are used as organisms in the process according to theinvention, they will be cultured, or grown, in the manner with which theskilled worker is familiar, depending on the host organism. As a rule,microorganisms will be grown in a liquid medium comprising a carbonsource, mostly in the form of sugars, a nitrogen source, mostly in theform of organic nitrogen sources such as yeast extract or salts such asammonium sulfate, trace elements such as iron, manganese and magnesiumsalts, and, if appropriate, vitamins, at temperatures between 0° C. and100° C., preferably between 10° C. to 60° C., while gassing in oxygen.During this process, the pH of the liquid nutrient may be kept constant,i.e. regulated during the culture period, or not. The culture can beeffected batchwise, semibatchwise or continuously. Nutrients can beintroduced at the beginning of the fermentation or fed insemicontinuously or continuously. The polyunsaturated fatty acidsproduced can be isolated from the organisms by methods with which theskilled worker is familiar, as described above; for example viaextraction, distillation, crystallization, if appropriate saltprecipitation and/or chromatography. To do so, the organisms canadvantageously be disrupted beforehand.

If the host organisms take the form of microorganisms, the processaccording to the invention is advantageously carried out at atemperature of between 0° C. to 95° C., preferably between 10° C. to 85°C., especially preferably between 15° C. to 75° C., very especiallypreferably between 15° C. to 45° C.

The pH is advantageously maintained at between pH 4 and pH 12,preferably between pH 6 and pH 9, especially preferably between pH 7 andpH 8.

The process according to the invention can be carried out batchwise,semibatchwise or continuously. A summary of known cultivation methods isto be found in the textbook by Chmiel (Bioprozeβtechnik 1. Einführung indie Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or inthe textbook by Storhas (Bioreaktoren and periphere Einrichtungen(Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

The culture medium to be used must satisfy in a suitable manner thedemands of the respective strains. There are descriptions of culturemedia for various microorganisms in the handbook “Manual of Methods forGeneral Bacteriology” of the American Society for Bacteriology(Washington D.C., USA, 1981).

These media which can be employed according to the invention usuallycomprise one or more carbon sources, nitrogen sources, inorganic salts,vitamins and/or trace elements, as described above.

Preferred carbon sources are sugars such as mono-, di- orpolysaccharides. Examples of very good carbon sources are glucose,fructose, mannose, galactose, ribose, sorbose, ribulose, lactose,maltose, sucrose, raffinose, starch or cellulose. Sugars can be put inthe media also via complex compounds such as molasses, or otherby-products of sugar refining. It may also be advantageous to addmixtures of various carbon sources. Other possible carbon sources areoils and fats such as, for example, soybean oil, sunflower oil, peanutoil and/or coconut fat, fatty acids such as, for example, palmitic acid,stearic acid and/or linoleic acid, alcohols and/or polyalcohols such as,for example, glycerol, methanol and/or ethanol and/or organic acids suchas, for example, acetic acid and/or lactic acid.

Nitrogen sources are usually organic or inorganic nitrogen compounds ormaterials comprising these compounds. Examples of nitrogen sourcesinclude ammonia gas, ammonia liquid or ammonium salts such as ammoniumsulfate, ammonium chloride, ammonium phosphate, ammonium carbonate orammonium nitrate, nitrates, urea, amino acids or complex nitrogensources such as corn steep liquor, soybean flour, soybean protein, yeastextract, meat extract and others. The nitrogen sources may be usedsingly or as mixtures.

Inorganic salt compounds which may be present in the media comprise thechloride, phosphoric or sulfate salts of calcium, magnesium, sodium,cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

For producing sulfur-containing fine chemicals, especially methionine,it is possible to use as sulfur source inorganic sulfur-containingcompounds such as, for example, sulfates, sulfites, dithionites,tetrathionates, thiosulfates, sulfides, but also organic sulfurcompounds such as mercaptans and thiols.

It is possible to use as phosphorus source phosphoric acid, potassiumdihydrogenphosphate or dipotassium hydrogenphosphate or thecorresponding sodium-containing salts.

Chelating agents can be added to the medium in order to keep the metalions in solution. Particularly suitable chelating agents comprisedihydroxyphenols such as catechol or protocatechuate, or organic acidssuch as citric acid.

The fermentation media employed according to the invention for theculture of microorganisms normally also comprise other growth factorssuch as vitamins or growth promoters, which include for example biotin,riboflavin, thiamine, folic acid, nicotinic acid, pantothenate andpyridoxine. Growth factors and salts are frequently derived from complexcomponents of the media, such as yeast extract, molasses, corn steepliquor and the like. Suitable precursors may also be added to theculture medium. The exact composition of the compounds in the mediadepends greatly on the particular experiment and will be decidedindividually for each specific case. Information on optimization ofmedia is obtainable from the textbook “Applied Microbiol. Physiology, APractical Approach” (editors P. M. Rhodes, P. F. Stanbury, IRL Press(1997) pp. 53-73, ISBN 0 19 963577 3). Growth media can also bepurchased from commercial suppliers, such as Standard 1 (Merck) or BHI(Brain heart infusion, DIFCO) and the like.

All the components of the media are sterilized either by heat (20 min at1.5 bar and 121° C.) or by filter sterilization. The components can besterilized either together or, if necessary, separately. All thecomponents of the media may be present at the start of culturing oroptionally be added continuously or batchwise.

The temperature of the culture is normally between 15° C. and 45° C.,preferably at 25° C. to 40° C., and can be kept constant or changedduring the experiment. The pH of the medium should be in the range from5 to 8.5, preferably around 7.0. The pH for the culturing can becontrolled during the culturing by adding basic compounds such as sodiumhydroxide, potassium hydroxide, ammonia or aqueous ammonia or acidiccompounds such as phosphoric acid or sulfuric acid. The development offoam can be controlled by employing antifoams such as, for example,fatty acid polyglycol esters. The stability of plasmids can bemaintained by adding to the medium suitable substances with a selectiveaction, such as, for example, antibiotics. Aerobic conditions aremaintained by introducing oxygen or oxygen-containing gas mixtures suchas, for example, ambient air into the culture. The temperature of theculture is normally 20° C. to 45° C., and preferably 25° C. to 40° C.The culture is continued until formation of the desired product is at amaximum. This aim is normally reached within 10 hours to 160 hours.

The dry matter content of the fermentation broths obtained in this wayand comprising in particular polyunsaturated fatty acids is normallyfrom 7.5 to 25% by weight.

The fermentation broth can then be processed further. Depending on therequirement, the biomass can be removed wholly or partly from thefermentation broth by separation methods such as, for example,centrifugation, filtration, decantation or a combination of thesemethods, or left completely in it. The biomass is advantageously workedup after removal.

However, the fermentation broth can also be thickened or concentrated byknown methods such as, for example, with the aid of a rotary evaporator,thin-film evaporator, falling-film evaporator, by reverse osmosis or bynanofiltration, without involving a cell removal step. This concentratedfermentation broth can then be worked up to obtain the fatty acidscomprised therein.

The fatty acids obtained in the process are also suitable as startingmaterial for the chemical synthesis of further products of value. Forexample, they can be used in combination with one another orindividually for the preparation of pharmaceuticals, foodstuffs, animalfeed or cosmetics.

The nucleic acid sequences which are used in the process and whichencode proteins with ω-3-desaturase activity are, alone or preferably incombination with further fatty acid biosynthesis genes, advantageouslyintroduced into an expression cassette (=nucleic acid construct) whichmakes possible the expression of the nucleic acids in an organism,advantageously a plant or a microorganism.

For the introduction, the nucleic acids used in the process areadvantageously subjected to amplification and ligation in the knownmanner. It is preferable to follow a procedure similar to the protocolof the Pfu-DNA polymerase or of a Pfu/Taq-DNA polymerase mixture. Theprimers are chosen to suit the sequence to be amplified. The primersshould expediently be chosen in such a way that the amplificatecomprises the entire codogenic sequence from the start codon to the stopcodon. After the amplification, it is expedient to analyze theamplificate. For example, it can be separated by gel electrophoresis andthen analyzed with regard to quality and quantity. Thereafter, theamplificate can be purified following a standard protocol (for exampleQiagen). An aliquot of the purified amplificate is now available for thesubsequent cloning step. Suitable cloning vectors are generally known tothe skilled worker. These include, in particular, vectors which arereplicable in microbial systems, that is to say mainly vectors whichensure efficient cloning in yeasts or fungi and which make possible thestable transformation of plants. Those which must be mentioned are, inparticular, various binary and co-integrated vector systems which aresuitable for the T-DNA-mediated transformation. Such vector systems are,as a rule, characterized in that they contain at least the vir genes,which are required for the Agrobacterium-mediated transformation, andthe sequences which delimit the T-DNA (T-DNA border). These vectorsystems preferably also comprise further cis-regulatory regions such aspromoters and terminators and/or selection markers with which suitabletransformed organisms can be identified. While co-integrated vectorsystems have vir genes and T-DNA sequences arranged on the same vector,binary systems are based on at least two vectors, one of which bears virgenes, but no T-DNA, while a second one bears T-DNA, but no vir gene. Asa consequence, the last-mentioned vectors are relatively small, easy tomanipulate and can be replicated both in E. coli and in Agrobacterium.These binary vectors include vectors from the pBIB-HYG, pPZP, pBecks,pGreen series. Preferably used in accordance with the invention areBin19, pBI101, pBinAR, pGPTV and pCAMBIA. An overview of binary vectorsand their use can be found in Hellens et al, Trends in Plant Science(2000) 5, 446-451. To prepare the vector, the vectors can first belinearized with the aid of restriction endonuclease(s) and then suitablymodified by enzymatic means. Thereafter, the vector is purified, and analiquot is employed for the cloning step. For the cloning, theenzymatically cleaved and, if appropriate, purified amplificate iscloned together with similarly prepared vector fragments, using ligase.Here, a specific nucleic acid construct, or vector or plasmid construct,may have one or else more than one codogenic gene segment. The codogenicgene segments in these constructs are preferably linked operably withregulatory sequences. The regulatory sequences include, in particular,plant sequences such as the above-described promoters and terminators.The constructs can advantageously be propagated stably under selectiveconditions in microorganisms, in particular Escherichia coli andAgrobacterium tumefaciens, and make possible the transfer ofheterologous DNA into plants or microorganisms.

With the advantageous use of cloning vectors, the nucleic acids used inthe process, the inventive nucleic acids and nucleic acid constructs canbe introduced into organisms such as microorganisms or, advantageously,plants, and thus be used in the transformation of plants, such as thosewhich are published, and cited, in: Plant Molecular Biology andBiotechnology (CRC Press, Boca Raton, Fla.), chapter 6/7, pp. 71-119(1993); F. F. White, Vectors for Gene Transfer in Higher Plants; in:Transgenic Plants, vol. 1, Engineering and Utilization, Ed.: Kung and R.Wu, Academic Press, 1993, 15-38; B. Jenes et al., Techniques for GeneTransfer, in: Transgenic Plants, vol. 1, Engineering and Utilization,Ed.: Kung and R. Wu, Academic Press (1993), 128-143; Potrykus, Annu.Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225. The nucleicacids used in the process, the inventive nucleic acids and nucleic acidconstructs and/or vectors, can thus be used for the recombinantmodification of a broad spectrum of organisms, advantageously plants, sothat the latter become better and/or more efficient PUFA producers.

A series of mechanisms exist which enable a modification of theω-3-desaturase protein according to the invention and of the furtherproteins used in the process, such as the Δ9-elongase, Δ6-desaturase,Δ8-desaturase, Δ6-elongase, Δ5-desaturase, Δ5-elongase or Δ4-desaturaseproteins, so that the yield, production and/or production efficiency ofthe advantageously polyunsaturated fatty acids in a plant, preferably inan oil crop or a microorganism, can be influenced directly as a resultof this modified protein. The number or activity of the Δ9-elongase,Δ6-desaturase, Δ8-desaturase, Δ6-elongase, Δ5-desaturase, Δ5-elongase,Δ4-desaturase or ω-3-desaturase proteins or genes can be increased sothat larger amounts of the gene products and thus ultimately largeramounts of the compounds of the general formula I are produced. Ade-novo synthesis in an organism which had lacked the activity andability to biosynthesize the compounds prior to the introduction of thegene(s) in question is also possible. The same applies to thecombination with further desaturases or elongases or further enzymesfrom the fatty acid and lipid metabolism. Also, the use of different,divergent sequences, i.e. sequences which differ at the DNA sequencelevel, may be advantageous, or the use of promoters for gene expressionwhich makes possible a different clock-dependent gene expression, forexample depending on the degree of maturity of a seed or oil-storingtissue.

Introducing a Δ9-elongase, Δ6-desaturase, Δ8-desaturase, Δ6-elongase,Δ5-desaturase, Δ5-elongase, Δ4-desaturase and/or ω-3-desaturase geneinto an organism alone or in combination with other genes into a cellmay not only increase the biosynthetic flux towards the end product, butalso increase the corresponding triacylglycerol composition or create itde novo. Likewise, the number or activity of other genes in the importof nutrients required for the biosynthesis of one or more fatty acids,oils, polar and/or neutral lipids may be increased, so that theconcentration of these precursors, cofactors or intermediates within thecells or within the storage compartment is increased, whereby theability of the cells to produce PUFAs is increased further, as describedhereinbelow. By optimizing the activity or increasing the number of oneor more Δ9-elongase, Δ6-desaturase, Δ8-desaturase, Δ6-elongase,Δ5-desaturase, Δ5-elongase, Δ4-desaturase or ω-3-desaturase genes whichare involved in the biosynthesis of these compounds, or by destroyingthe activity of one or more genes which are involved in breaking downthese compounds, it may be possible to increase the yield, productionand/or production efficiency of fatty acid and lipid molecules fromorganisms and advantageously from plants.

The isolated nucleic acid molecules used in the process according to theinvention encode proteins or parts of these, the proteins or theindividual protein or parts thereof comprising an amino acid sequencewith sufficient homology with an amino acid sequence which is shown inthe sequences SEQ ID NO: 2 so that the proteins or parts thereof retaina ω-3-desaturase activity. The proteins or parts thereof, which is/areencoded by the nucleic acid molecule(s), preferably still retain theiressential enzymatic activity and the ability of participating in themetabolism of compounds required in the formation of cell membranes orlipid bodies in organisms, advantageously in plants, or in the transportof molecules across these membranes. Advantageously, the proteinsencoded by the nucleic acid molecules have at least approximately 60%,preferably at least approximately 70% and more preferably at leastapproximately 80% or 90% and most preferably at least approximately 95%,96%, 97%, 98%, 99% or more identity with the amino acid sequence shownin SEQ ID NO: 2. For the purposes of the invention, homology orhomologous is understood as meaning identity or identical.

The homology was calculated over the entire amino acid or nucleic acidsequence region. A series of programs which are based on the variousalgorithms are available to those skilled in the art for comparingdifferent sequences. In this context, the algorithms of Needleman andWunsch or Smith and Waterman give especially reliable results. To carryout the sequence alignments, the program PileUp (J. Mol. Evolution., 25,351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153) or the programsGap and BestFit [Needleman and Wunsch (J. Mol. Biol. 48; 443-453 (1970))and Smith and Waterman (Adv. Appl. Math. 2; 482-489 (1981)), which arepart of the GCG software packet [Genetics Computer Group, 575 ScienceDrive, Madison, Wis., USA 53711 (1991)], were used. The sequencehomology values stated above as percentages were determined over theentire sequence region using the program GAP, with the followingsettings: Gap Weight: 50, Length Weight: 3, Average Match: 10.000 andAverage Mismatch: 0.000. Unless otherwise specified, these settings werealways used as standard settings for sequence alignments.

Essential enzymatic activity of the ω-3-desaturase used in the processaccording to the invention is understood as meaning that, in comparisonwith the proteins/enzymes encoded by the sequence SEQ ID NO: 1 and theirderivatives, it retain at least an enzymatic activity of at least 10%,preferably 20%, especially preferably 30% and very especially 40% andcan thus participate in the metabolism of compounds required in thesynthesis of fatty acids, fatty acid esters such as diacylglyceridesand/or triacylglycerides in an organism, advantageously a plant or plantcell, or in the transport of molecules across membranes, meaning C₁₈-,C₂₀- or C₂₂-carbon chains in the fatty acid molecule with double bondsat least two, advantageously three, four, five or six positions.

Nucleic acids which can be used advantageously in the process arederived from bacteria, fungi, diatoms, animals such as Caenorhabditis orplants such as algae or mosses, such as the genera Shewanella,Physcomitrella, Thraustochytrium, Fusarium, Phytophthora, Ceratodon,Isochrysis, Aleurita, Muscarioides, Mortierella, Phaeodactylum,Cryphthecodinium, specifically from the genera and species Thallasiosirapseudonona, Euglena gracilis, Physcomitrella patens, Phytophtorainfestans, Fusarium graminaeum, Cryptocodinium cohnii, Ceratodonpurpureus, Isochrysis galbana, Aleurita farinosa, Thraustochytrium sp.,Muscarioides viallii, Mortierella alpina, Phaeodactylum tricornutum orCaenorhabditis elegans or especially advantageously Phytophtorainfestans, Thallasiosira pseudonona or Cryptocodinium cohnii.

As an alternative, it is possible to use, in the process according tothe invention, nucleotide sequences which encode ω-3-desaturase andwhich hybridize, advantageously under stringent conditions, with anucleotide sequence as shown in SEQ ID NO: 1.

The nucleic acid sequences used in the process are advantageouslyintroduced in an expression cassette which enables the expression of thenucleic acids in organisms such as microorganisms or plants.

In this context, the nucleic acid sequences which encode theω-3-desaturase are advantageously linked functionally with one or moreregulatory signals to increase gene expression. These regulatorysequences should enable the targeted expression of the genes and proteinexpression. For example, this may mean, depending on the host organism,that the gene is expressed and/or overexpressed only after induction hastaken place, or else that it is expressed and/or overexpressedimmediately. For example, these regulatory sequences take the form ofsequences to which inductors or repressors bind and thus regulate theexpression of the nucleic acid. In addition to these new regulatorysequences, or instead of these sequences, the natural regulation ofthese sequences may still be present before the actual structural genesand, if appropriate, may have been genetically modified in such a waythat the natural regulation has been switched off and the expression ofthe genes enhanced. The expression cassette (=expression construct=geneconstruct) may, however, also be simpler in construction, that is to sayno additional regulatory signals were inserted before the nucleic acidsequence or its derivatives, and the natural promoter together with itsregulation was not removed. Instead, the natural regulatory sequence wasmutated in such a way that regulation no longer takes place and/or geneexpression is enhanced. These modified promoters can be placed beforethe natural gene in order to increase the activity either in the form ofpart-sequences (=promoter with parts of the nucleic acid sequencesaccording to the invention) or else alone. Moreover, the gene constructcan advantageously also comprise one or more what are known as “enhancersequences” in functional linkage with the promoter, and these enable anincreased expression of the nucleic acid sequence. Also, it is possibleto insert additional advantageous sequences at the 3′ end of the DNAsequences, such as further regulatory elements or terminators. Theω-3-desaturase genes can be present in the expression cassette (=geneconstruct) as one or more copies. The same applies to the other fattyacid biosynthesis genes which are used in combination with theω-3-desaturase according to the invention. Advantageously, only in eachcase one copy of the genes is present in the expression cassette. Thisgene construct, or the gene constructs, can be expressed together in thehost organism. In this context, the gene construct(s) can be inserted inone or more vectors and be present in the cell in free form or elseinserted in the genome. It is advantageous for the insertion of furthergenes in the host genome when the genes to be expressed are presenttogether in one gene construct.

In this context, the regulatory sequences or factors can, as describedabove, preferably have a positive effect on the gene expression of thegenes which have been introduced, thus increasing it. Thus, enhancementof the regulatory elements can advantageously take place at thetranscription level by using strong transcription signals such aspromoters and/or enhancers. Besides, however, an enhancement of thetranslation is also possible, for example by improving the stability ofthe mRNA.

A further embodiment of the invention are one or more gene constructswhich comprise one or more sequences which are defined by SEQ ID NO: 1or its derivatives and code for polypeptides according to SEQ ID NO: 2.The abovementioned ω-3-desaturase proteins advantageously result in adesaturation of ω-6-fatty acids, the substrate advantageously havingtwo, three, four or five double bonds and advantageously 18, 20 or 22carbon atoms in the fatty acid molecule. The same applies to theirhomologs, derivatives or analogs which are linked operably with one ormore regulatory signals, advantageously for increasing gene expression.

Advantageous regulatory sequences for the novel process are present forexample in promoters such as the cos, tac, trp, tet, trp-tet, lpp, lac,lpp-lac, lacIq, T7, T5, T3, gal, trc, ara, SP6, λ-PR or λ-PL promoterand are advantageously used in Gram-negative bacteria. Furtheradvantageous regulatory sequences are present for example in theGram-positive promoters amy and SPO2, in the yeast or fungal promotersADC1, MFα, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH or in the plantpromoters CaMV/35S [Franck et al., Cell 21 (1980) 285-294], PRP1 [Wardet al., Plant. Mol. Biol. 22 (1993)], SSU, OCS, lib4, usp, STLS1, B33,nos or in the ubiquitin or phaseolin promoter. Also advantageous in thiscontext are inducible promoters, such as the promoters described inEP-A-0 388 186 (benzylsulfonamide-inducible), Plant J. 2, 1992:397-404(Gatz et al., tetracyclin-inducible), EP-A-0 335 528(abscisic-acid-inducible) or WO 93/21334 (ethanol- orcyclohexenol-inducible). Further suitable plant promoters are thepromoter of cytosolic FBPase or the ST-LSI promoter from potato(Stockhaus et al., EMBO J. 8, 1989, 2445), thephosphoribosyl-pyrophosphate amidotransferase promoter from Glycine max(Genbank accession No. U87999) or the node-specific promoter describedin EP-A-0 249 676. Especially advantageous promoters are promoters whichenable the expression in tissues which are involved in the biosynthesisof fatty acids. Very especially advantageous are seed-specific promoterssuch as the USP promoter in accordance with the practice, but also otherpromoters such as the LeB4, DC3, phaseolin or napin promoters. Furtherespecially advantageous promoters are seed-specific promoters which canbe used for monocotyledonous or dicotyledonous plants and which aredescribed in U.S. Pat. No. 5,608,152 (napin promoter from oilseed rape),WO 98/45461 (oleosin promoter from Arobidopsis, U.S. Pat. No. 5,504,200(phaseolin promoter from Phaseolus vulgaris), WO 91/13980 (Bce4 promoterfrom Brassica), by Baeumlein et al., Plant J., 2, 2, 1992:233-239 (LeB4promoter from a legume), these promoters being suitable for dicots. Thefollowing promoters are suitable for example for monocots: lpt-2 orlpt-1 promoter from barley (WO 95/15389 and WO 95/23230), hordeinpromoter from barley and other promoters which are suitable and whichare described in WO 99/16890.

In principle, it is possible to use all natural promoters together withtheir regulatory sequences, such as those mentioned above, for the novelprocess. Likewise, it is possible and advantageous to use syntheticpromoters, either additionally or alone, especially when they mediate aseed-specific expression, such as, for example, as described in WO99/16890.

To obtain a particularly high PUFA content especially in transgenicplants, the ω-3-desaturase and/or PUFA biosynthesis genes shouldadvantageously be expressed in a seed-specific manner in oilseed crops.To this end, it is possible to use seed-specific promoters or thosepromoters which are active in the embryo and/or in the endosperm. Inprinciple, seed-specific promoters can be isolated both fromdicotyledonous and from monocotyledonous plants. Advantageous preferredpromoters are listed hereinbelow: USP (=unknown seed protein) andvicilin (Vicia faba) [Baumlein et al., Mol. Gen. Genet., 1991, 225(3)],napin (oilseed rape) [U.S. Pat. No. 5,608,152], acyl carrier protein(oilseed rape) [U.S. Pat. No. 5,315,001 and WO 92/18634], oleosin(Arabidopsis thaliana) [WO 98/45461 and WO 93/20216], phaseolin(Phaseolus vulgaris) [U.S. Pat. No. 5,504,200], Bce4 [WO 91/13980],Legume B4 (LegB4 promoter) [Baumlein et al., Plant J., 2,2, 1992], Lpt2and lpt1 (barley) [WO 95/15389 and WO95/23230], seed-specific promotersfrom rice, maize and wheat [WO 99/16890], Amy32b, Amy 6-6 and aleurain[U.S. Pat. No. 5,677,474], Bce4 (oilseed rape) [U.S. Pat. No.5,530,149], glycinin (soybean) [EP 571 741], phosphoenol-pyruvatecarboxylase (soybean) [JP 06/62870], ADR12-2 (soybean) [WO 98/08962],isocitrate lyase (oilseed rape) [U.S. Pat. No. 5,689,040] or α-amylase(barley) [EP 781 849].

The expression of plant genes can also be facilitated via a chemicallyinducible promoter (for a review see Gatz 1997, Annu. Rev. PlantPhysiol. Plant Mol. Biol., 48:89-108). Chemically inducible promotersare particularly suitable when it is desired that gene expression iseffected in a clock-controlled manner. Examples of such promoters aresalicylic-acid-inducible promoter (WO 95/19443), a tetracyclin-induciblepromoter (Gatz et al. (1992) Plant J. 2, 397-404) and anethanol-inducible promoter.

To ensure a stable integration of the biosynthetic genes into thetransgenic plant over a plurality of generation, each of the nucleicacids used in the process which encode the ω-3-desaturase gene orfurther fatty acid biosynthesis genes such as Δ9-elongase,Δ6-desaturase, Δ8-desaturase, Δ6-elongase, Δ5-desaturase, Δ5-elongaseand/or Δ4-desaturase should be expressed under the control of a separatepromoter, preferably a different promoter, since repeating sequencemotifs can lead to instability of the T-DNA, or to recombination events.In this context, the expression cassette is advantageously constructedin such a way that a promoter is followed by a suitable cleavage site,advantageously in a polylinker, for insertion of the nucleic acid to beexpressed and then, if appropriate, a terminator is positioned behindthe polylinker. This sequence is repeated several times, preferablythree, four or five times, so that up to five genes can be combined inone construct and introduced into the transgenic plant in order to beexpressed. Advantageously, the sequence is repeated up to three times.To express the nucleic acid sequences, the latter are inserted behindthe promoter via a suitable cleavage site, for example in thepolylinker. Advantageously, each nucleic acid sequence has its ownpromoter and, if appropriate, its own terminator. Such advantageousconstructs are disclosed, for example, in DE 101 02 337 or DE 101 02 338(see, for example, in the appended sequence listings). However, it isalso possible to insert a plurality of nucleic acid sequences behind apromoter and, if appropriate, before a terminator. Here, the insertionsite, or the sequence, of the inserted nucleic acids in the expressioncassette is not of critical importance, that is to say a nucleic acidsequence can be inserted at the first or last position in the cassettewithout its expression being substantially influenced thereby.Advantageously, different promoters such as, for example, the USP, LegB4or DC3 promoter, and different terminator can be used in the expressioncassette. However, it is also possible to use only one type of promoterin the cassette, which, however, may lead to undesired recombinationevents.

As described above, the transcription of the genes which have beenintroduced should advantageously be terminated by suitable terminatorsat the 3′ end of the biosynthesis genes which have been introduced(behind the stop codon). An example of a sequence which can be used inthis context is the OCS1 terminator. As is the case with the promotersas well, different terminator sequences should be used for each gene.

As described above, the gene construct can also comprise further genesto be introduced into the organisms. It is possible and advantageous tointroduce into the host organisms, and to express therein, regulatorygenes such as genes for inductors, repressors or enzymes which, owing totheir enzyme activity, engage in the regulation of one or more genes ofa biosynthetic pathway. These genes can be of heterologous or ofhomologous origin. Moreover, further biosynthesis genes of the fattyacid or lipid metabolism can advantageously be present in the nucleicacid construct, or gene construct; however, these genes can also bepresent on one or more further nucleic acid constructs. A biosynthesisgene of the fatty acid or lipid metabolism which is preferably chosen isa gene from the group consisting of acyl-CoA dehydrogenase(s), acyl-ACP[=acyl carrier protein] desaturase(s), acyl-ACP thioesterase(s), fattyacid acyltransferase(s), acyl-CoA:lysophospholipid acyltransferase(s),fatty acid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme Acarboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s),fatty acid acetylenase(s), lipoxygenase(s), triacylglycerol lipase(s),allenoxide synthase(s), hydroperoxide lyase(s) or fatty acid elongase(s)or combinations thereof. Especially advantageous nucleic acid sequencesare biosynthesis genes of the fatty acid or lipid metabolism selectedfrom the group of the acyl-CoA:lysophospholipid acyltransferase,Δ4-desaturase, Δ5-desaturase, Δ6-desaturase, Δ8-desaturase,Δ9-desaturase, Δ12-desaturase, Δ5-elongase, Δ6-elongase and/orΔ9-elongase.

In this context, the abovementioned nucleic acids or genes can be clonedinto expression cassettes, like those mentioned above, in combinationwith other elongases and desaturases and used for transforming plantswith the aid of Agrobacterium.

Here, the regulatory sequences or factors can, as described above,preferably have a positive effect on, and thus enhance, the geneexpression of the genes which have been introduced. Thus, enhancement ofthe regulatory elements can advantageously take place at thetranscriptional level by using strong transcription signals such aspromoters and/or enhancers. However, an enhanced translation is alsopossible, for example by improving the stability of the mRNA. Inprinciple, the expression cassettes can be used directly forintroduction into the plants or else be introduced into a vector.

These advantageous vectors, preferably expression vectors, comprise thenucleic acids according to the invention which encode ω-3-desaturases,which acids are used in the process, and, if appropriate, furthernucleic acids which are used in the process and which encodeΔ9-elongases, Δ6-desaturases, Δ8-desaturases, Δ6-elongases,Δ5-desaturases, Δ5-elongases or Δ4-desaturases or else a nucleic acidconstruct which comprises the nucleic acid used either alone or incombination with further biosynthesis genes of the fatty acid or lipidmetabolism such as the acyl-CoA:lysophospholipid acyltransferases,Δ4-desaturases, Δ5-desaturases, Δ6-desaturases, Δ8-desaturases,Δ9-desaturases, Δ12-desaturases, ω-3-desaturases, Δ5-elongases,Δ6-elongases and/or Δ9-elongases. As used in the present context, theterm “vector” refers to a nucleic acid molecule which is capable oftransporting another nucleic acid to which it is bound. One type ofvector is a “plasmid”, a circular double-stranded DNA loop into whichadditional DNA segments can be ligated. A further type of vector is aviral vector, it being possible for additional DNA segments to beligated into the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they have been introduced (forexample bacterial vectors with bacterial replication origin). Othervectors are advantageously integrated into the genome of a host cellwhen they are introduced into the host cell, and thus replicate togetherwith the host genome. Moreover, certain vectors can govern theexpression of genes with which they are in operable linkage. Thesevectors are referred to in the present context as “expression vectors”.Usually, expression vectors which are suitable for DNA recombinationtechniques take the form of plasmids. In the present description,“plasmid” and “vector” can be used exchangeably since the plasmid is theform of vector which is most frequently used. However, the invention isalso intended to cover other forms of expression vectors, such as viralvectors, which exert similar functions. Furthermore, the term “vector”is also intended to encompass other vectors with which the skilledworker is familiar, such as phages, viruses such as SV40, CMV, TMV,transposons, IS elements, phasmids, phagemids, cosmids, linear orcircular DNA.

The recombinant expression vectors advantageously used in the processcomprise the above described nucleic acid sequences and/or the abovedescribed gene construct in a form which is suitable for expressing thenucleic acids used in a host cell, which means that the recombinantexpression vectors comprises one or more regulatory sequences, selectedon the basis of the host cells used for the expression, which regulatorysequence(s) is/are linked operably with the nucleic acid sequence to beexpressed. In a recombinant expression vector, “linked operably” meansthat the nucleotide sequence of interest is bound to the regulatorysequence(s) in such a way that the expression of the nucleotide sequenceis possible and they are bound to each other in such a way that bothsequences carry out the predicted function which is ascribed to thesequence (for example in an in-vitro transcription/translation system,or in a host cell if the vector is introduced into the host cell). Theterm “regulatory sequence” is intended to comprise promoters, enhancersand other expression control elements (for example polyadenylationsignals). These regulatory sequences are described, for example, inGoeddel: Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990), or see: Gruber and Crosby, in: Methodsin Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton,Fla., Ed.: Glick and Thompson, Chapter 7, 89-108, including thereferences cited therein. Regulatory sequences comprise those whichgovern the constitutive expression of a nucleotide sequence in manytypes of host cell and those which govern the direct expression of thenucleotide sequence only in specific host cells under specificconditions. The skilled worker knows that the design of the expressionvector can depend on factors such as the choice of host cell to betransformed, the desired expression level of the protein and the like.

The recombinant expression vectors used can be designed for expressingω-3-desaturases, Δ9-elongases, Δ6-desaturases, Δ8-desaturases,Δ6-elongases, Δ5-desaturases, Δ5-elongases and/or Δ4-desaturases inprokaryotic or eukaryotic cells. This is advantageous since, for thesake of simplicity, intermediate steps of the vector construction arefrequently carried out in microorganisms. For example, theω-3-desaturase, Δ9-elongase, Δ6-desaturase, Δ8-desaturase, Δ6-elongase,Δ5-desaturase, Δ5-elongase and/or Δ4-desaturase genes can be expressedin bacterial cells, insect cells (using baculovirus expression vectors),yeast cells and other fungal cells (see Romanos, M. A., et al. (1992)“Foreign gene expression in yeast: a review”, Yeast 8:423-488; van denHondel, C. A. M. J. J., et al. (1991) “Heterologous gene expression infilamentous fungi”, in: More Gene Manipulations in Fungi, J. W. Bennet &L. L. Lasure, Ed., pp. 396-428: Academic Press: San Diego; and van denHondel, C. A. M. J. J., & Punt, P. J. (1991) “Gene transfer systems andvector development for filamentous fungi, in: Applied Molecular Geneticsof Fungi, Peberdy, J. F., et al., Ed., pp. 1-28, Cambridge UniversityPress: Cambridge), algae (Falciatore et al., 1999, Marine Biotechnology.1, 3:239-251), ciliates of the types: Holotrichia, Peritrichia,Spirotrichia, Suctoria, Tetrahymena, Paramecium, Colpidium, Glaucoma,Platyophrya, Potomacus, Desaturaseudocohnilembus, Euplotes,Engelmaniella and Stylonychia, in particular the genus Stylonychialemnae, using vectors following a transformation process as described inWO 98/01572, and preferably in cells of multi-celled plants (seeSchmidt, R. and Willmitzer, L. (1988) “High efficiency Agrobacteriumtumefaciens-mediated transformation of Arabidopsis thaliana leaf andcotyledon explants” Plant Cell Rep.: 583-586; Plant Molecular Biologyand Biotechnology, C Press, Boca Raton, Fla., chapter 6/7, pp. 71-119(1993); F. F. White, B. Jenes et al., Techniques for Gene Transfer, in:Transgenic Plants, Vol. 1, Engineering and Utilization, Ed.: Kung and R.Wu, Academic Press (1993), 128-43; Potrykus, Annu. Rev. Plant Physiol.Plant Molec. Biol. 42 (1991), 205-225 (and references cited therein)).Suitable host cells are furthermore discussed in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). As an alternative, the recombinant expressionvector can be transcribed and translated in vitro, for example usingT7-promoter regulatory sequences and T7-polymerase.

In most cases, the expression of proteins in prokaryotes is performedusing vectors comprising constitutive or inducible promoters whichcontrol the expression of fusion or nonfusion proteins. Examples oftypical fusion expression vectors are pGEX (Pharmacia Biotech Inc;Smith, D. B., and Johnson, K. S. (1988) Gene 67:31-40), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway,N.J.), where glutathione S-transferase (GST), maltose E-binding proteinand protein A, respectively, are fused with the recombinant targetprotein.

Examples of suitable inducible nonfusion E. coli expression vectors are,inter alia, pTrc (Amann et al. (1988) Gene 69:301-315) and pET 11d(Studier et al., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 60-89). The target geneexpression of the pTrc vector is based on the transcription from ahybrid trp-lac fusion promoter by host RNA polymerase. The target geneexpression from the pET 11d vector is based on the transcription of aT7-gn10-lac fusion promoter, which is mediated by a coexpressed viralRNA polymerase (T7 gn1). This viral polymerase is provided by the hoststrains BL21 (DE3) or HMS174 (DE3) from a resident λ-prophage whichharbors a T7 gn1 gene under the transcriptional control of the lacUV 5promoter.

The skilled worker is familiar with other vectors which are suitable inprokaryotic organisms; these vectors are, for example, in E. coli,pLG338, pACYC184, the pBR series such as pBR322, the pUC series such aspUC18 or pUC19, the M113 mp series, pKC30, pRep4, pHS1, pHS2, pPLc236,pMBL24, pLG200, pUR290, pIN-III113-B1, λgt11 or pBdCl, in StreptomycespIJ101, pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or pBD214,in Corynebacterium pSA77 or pAJ667.

In a further embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in the yeast S. cerevisiaecomprise pYeDesaturasec1 (Baldari et al. (1987) Embo J. 6:229-234), pMFa(Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al.(1987) Gene 54:113-123) and pYES2 (Invitrogen Corporation, San Diego,Calif.). Vectors and processes for the construction of vectors which aresuitable for use in other fungi, such as the filamentous fungi, comprisethose which are described in detail in: van den Hondel, C. A. M. J. J.,& Punt, P. J. (1991) “Gene transfer systems and vector development forfilamentous fungi, in: Applied Molecular Genetics of fungi, J. F.Peberdy et al., Ed., pp. 1-28, Cambridge University Press: Cambridge, orin: More Gene Manipulations in Fungi [J. W. Bennett & L. L. Lasure, Ed.,pp. 396-428: Academic Press: San Diego]. Further suitable yeast vectorsare, for example, pAG-1, YEp6, YEp13 or pEMBLYe23.

As an alternative, the ω-3-desaturases, Δ9-elongases, Δ6-desaturases,Δ8-desaturases, Δ6-elongases, Δ5-desaturases, Δ5-elongases and/orΔ4-desaturases can be expressed in insect cells using baculovirusexpression vectors. Baculovirus vectors which are available for theexpression of proteins in cultured insect cells (for example Sf9 cells)comprise the pAc series (Smith et al. (1983) Mol. Cell. Biol.3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology170:31-39).

The abovementioned vectors are only a small overview of possiblesuitable vectors. Further plasmids are known to the skilled worker andare described, for example, in: Cloning Vectors (Ed., Pouwels, P. H., etal., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). Forfurther suitable expression systems for prokaryotic and eukaryoticcells, see the chapters 16 and 17 of Sambrook, J., Fritsch, E. F., andManiatis, T., Molecular Cloning: A Laboratory Manual, 2^(nd) edition,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

In a further embodiment of the process, the ω-3-desaturases,Δ9-elongases, Δ6-desaturases, Δ8-desaturases, Δ6-elongases,Δ5-desaturases, Δ5-elongases and/or Δ4-desaturases can be expressed insingle-cell plant cells (such as algae), see Falciatore et al., 1999,Marine Biotechnology 1 (3):239-251 and the references cited therein, andplant cells from higher plants (for example Spermatophytes, such asarable crops). Examples of plant expression vectors comprise those whichare described in detail in: Becker, D., Kemper, E., Schell, J., andMasterson, R. (1992) “New plant binary vectors with selectable markerslocated proximal to the left border”, Plant Mol. Biol. 20:1195-1197; andBevan, M. W. (1984) “Binary Agrobacterium vectors for planttransformation”, Nucl. Acids Res. 12:8711-8721; Vectors for GeneTransfer in Higher Plants; in: Transgenic Plants, Vol. 1, Engineeringand Utilization, Ed.: Kung and R. Wu, Academic Press, 1993, p. 15-38.

A plant expression cassette preferably comprises regulatory sequenceswhich are capable of controlling the gene expression in plant cells andwhich are functionally linked so that each sequence can fulfill itsfunction, such as transcriptional termination, for examplepolyadenylation signals. Preferred polyadenylation signals are thosewhich are derived from Agrobacterium tumefaciens T-DNA, such as the gene3 of the Ti plasmid pTiACH₅, which is known as octopine synthase (Gielenet al., EMBO J. 3 (1984) 835 et seq.) or functional equivalents ofthese, but all other terminators which are functionally active in plantsare also suitable.

Since plant gene expression is very often not limited to transcriptionallevels, a plant expression cassette preferably comprises otherfunctionally linked sequences such as translation enhancers, for examplethe overdrive sequence, which comprises the 5′-untranslated tobaccomosaic virus leader sequence, which increases the protein/RNA ratio(Gallie et al., 1987, Nucl. Acids Research 15:8693-8711).

As described above, plant gene expression must be functionally linked toa suitable promoter which performs the expression of the gene in atimely, cell-specific or tissue-specific manner. Promoters which can beused are constitutive promoters (Benfey et al., EMBO J. 8 (1989)2195-2202) such as those which are derived from plant viruses such as35S CAMV (Franck et al., Cell 21 (1980) 285-294), 19S CaMV (see alsoU.S. Pat. No. 5,352,605 and WO 84/02913) or plant promoters such as thepromoter of the Rubisco small subunit, which is described in U.S. Pat.No. 4,962,028.

Other preferred sequences for the use in functional linkage in plantgene expression cassettes are targeting sequences which are required fortargeting the gene product into its relevant cell compartment (for areview, see Kermode, Crit. Rev. Plant Sci. 15, 4 (1996) 285-423 andreferences cited therein), for example into the vacuole, the nucleus,all types of plastids, such as amyloplasts, chloroplasts, chromoplasts,the extracellular space, the mitochondria, the endoplasmic reticulum,oil bodies, peroxisomes and other compartments of plant cells.

As described above, plant gene expression can also be facilitated via achemically inducible promoter (for a review, see Gatz 1997, Annu. Rev.Plant Physiol. Plant Mol. Biol., 48:89-108). Chemically induciblepromoters are particularly suitable if it is desired that genes areexpressed in a time-specific manner. Examples of such promoters are asalicylic-acid-inducible promoter (WO 95/19443), a tetracyclin-induciblepromoter (Gatz et al. (1992) Plant J. 2, 397-404) and anethanol-inducible promoter.

Promoters which respond to biotic or abiotic stress conditions are alsosuitable promoters, for example the pathogen-induced PRP1-gene promoter(Ward et al., Plant Mol. Biol. 22 (1993) 361-366), the heat-induciblehsp80 promoter from tomato (U.S. Pat. No. 5,187,267), the cold-induciblealpha-amylase promoter from potato (WO 96/12814) or the wound-induciblepinII promoter (EP-A-0 375 091).

The promoters which are especially preferred are those which bring aboutthe expression of genes in tissues and organs in which fatty acid, lipidand oil biosynthesis takes place, in seed cells such as the cells ofendosperm and of the developing embryo. Suitable promoters are the napingene promoter from oilseed rape (U.S. Pat. No. 5,608,152), the USPpromoter from Vicia faba (Baeumlein et al., Mol. Gen. Genet., 1991, 225(3):459-67), the oleosin promoter from Arabidopsis (WO 98/45461), thephaseolin promoter from Phaseolus vulgaris (U.S. Pat. No. 5,504,200),the Bce4 promoter from Brassica (WO 91/13980) or the legumin B4 promoter(LeB4; Baeumlein et al., 1992, Plant Journal, 2 (2):233-9), andpromoters which bring about the seed-specific expression inmonocotyledonous plants such as maize, barley, wheat, rye, rice and thelike. Suitable promoters to be taken into consideration are the lpt2 orlpt1 gene promoter from barley (WO 95/15389 and WO 95/23230) or thosewhich are described in WO 99/16890 (promoters from the barley hordeingene, the rice glutelin gene, the rice oryzin gene, the rice prolamingene, the wheat gliadin gene, wheat glutelin gene, the maize zein gene,the oat glutelin gene, the sorghum kasirin gene, the rye secalin gene).

In particular, the multiparallel expression of the ω-3-desaturases,Δ9-elongases, Δ6-desaturases, Δ8-desaturases, Δ6-elongases,Δ5-desaturases, Δ5-elongases and/or Δ4-desaturases used in the processmay be desired. Such expression cassettes can be introduced via asimultaneous transformation of a plurality of individual expressionconstructs or, preferably, by combining a plurality of expressioncassettes on one construct. Also, it is possible to transform aplurality of vectors with in each case a plurality of expressioncassettes and to transfer them to the host cell.

Likewise especially suitable are promoters which bring about theplastid-specific expression since plastids are the compartment in whichthe precursors and some end products of lipid biosynthesis aresynthesized. Suitable promoters such as the viral RNA-polymerasepromoter, are described in WO 95/16783 and WO 97/06250, and the clpPpromoter from Arabidopsis, described in WO 99/46394.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. The terms“transformation” and “transfection”, conjugation and transduction, asused in the present context, are intended to comprise a multiplicity ofprior-art processes for introducing foreign nucleic acid (for exampleDNA) into a host cell, including calcium phosphate or calcium chloridecoprecipitation, DEAE-dextran-mediated transfection, lipofection,natural competence, chemically mediated transfer, electroporation orparticle bombardment. Suitable methods for the transformation ortransfection of host cells, including plant cells, can be found inSambrook et al. (Molecular Cloning: A Laboratory Manual, 2^(nd) ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989) and other laboratory manuals, such as Methodsin Molecular Biology, 1995, Vol. 44, Agrobacterium protocols, Ed.:Gartland and Davey, Humana Press, Totowa, N.J.

Host cells which are capable, in principle, of taking up the nucleicacid according to the invention, the gene product according to theinvention or the vector according to the invention are all prokaryoticor eukaryotic organisms. The host organisms which are advantageouslyused are microorganisms such as fungi or yeasts, or plant cells,preferably plants or parts thereof. Fungi, yeasts or plants are used bypreference, especially preferably plants, very especially preferablyplants such as oil crops which comprise large amounts of lipidcompounds, such as oilseed rape, evening primrose, hemp, thistle,peanut, canola, linseed, soybean, safflower, sunflower, borage, orplants such as maize, wheat, rye, oats, triticale, rice, barley, cotton,cassaya, pepper, Tagetes, Solanaceae plants such as potato, tobacco,eggplant and tomato, Vicia species, pea, alfalfa, bushy plants (coffee,cacao, tea), Salix species, trees (oil palm, coconut) and perennialgrasses and fodder crops. Especially preferred plants according to theinvention are oil crops such as soybean, peanut, oilseed rape, canola,linseed, hemp, evening primrose, sunflower, safflower, trees (oil palm,coconut).

As described above, a further subject matter according to the inventionare an isolated nucleic acid sequence which encodes polypeptides withω-3-desaturase activity where the ω-3-desaturases encoded by the nucleicacid sequences converts C₁₈-, C₂₀- and C₂₂-fatty acids with two, three,four or five double bonds and advantageously polyunsaturated C₁₈-fattyacids with two or three double bonds and polyunsaturated C₂₀-fatty acidswith two, three or four double bonds. C₂₂-Fatty acids with four or fivedouble bonds are also desaturated.

In an advantageous embodiment, the term “nucleic acid (molecule)” asused in the present text additionally comprises the untranslatedsequence at the 3′ and at the 5′ terminus of the coding gene region: atleast 500, preferably 200, especially preferably 100 nucleotides of thesequence upstream of the 5′ terminus of the coding region and at least100, preferably 50, especially preferably 20 nucleotides of the sequencedownstream of the 3′ terminus of the coding gene region. An “isolated”nucleic acid molecule is separated from other nucleic acid moleculeswhich are present in the natural source of the nucleic acid. An“isolated” nucleic acid preferably has no sequences which naturallyflank the nucleic acid in the genomic DNA of the organism from which thenucleic acid is derived (for example sequences which are located at the5′ and 3′ termini of the nucleic acid). In various embodiments, theisolated ω-3-desaturase molecule can, for example, comprise less thanapproximately 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb ofnucleotide sequences which naturally flank the nucleic acid molecule inthe genomic DNA of the cell from which the nucleic acid is derived.

The nucleic acid molecules used in the process, for example a nucleicacid molecule with a nucleotide sequence of SEQ ID NO: 1 or partthereof, can be isolated using standard techniques of molecular biologyand the sequence information provided herein. Also, for example ahomologous sequence or homologous, conserved sequence regions at the DNAor amino acid level can be identified with the aid of comparativealgorithms. These sequence regions can be used as hybridization probeand standard hybridization techniques (such as, for example, describedin Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989) for isolating further nucleic acid sequenceswhich are useful in the process. Moreover, a nucleic acid moleculecomprising a complete sequence of SEQ ID NO: 1 or part thereof can beisolated by polymerase chain reaction, where oligonucleotide primerswhich on the basis of this sequence or parts thereof are used (forexample, a nucleic acid molecule comprising the complete sequence orpart thereof can be isolated by polymerase chain reaction usingoligonucleotide primers which have been generated on the basis of thisvery sequence). For example, mRNA can be isolated from cells (forexample by the guanidinium thiocyanate extraction process by Chirgwin etal. (1979) Biochemistry 18:5294-5299) and cDNA can be generated by meansof reverse transcriptase (for example Moloney-MLV reverse transcriptase,from Gibco/BRL, Bethesda, Md., or AMV reverse transcriptase, fromSeikagaku America, Inc., St. Petersburg, Fla.). Syntheticoligonucleotide primers for the amplification by means of polymerasechain reaction can be generated on the basis of the sequence shown inSEQ ID NO: 1 or with the aid of the amino acid sequence shown in SEQ IDNO: 2. In accordance with the invention a nucleic acid can be amplifiedin accordance with standard PCR amplification techniques using cDNA or,alternatively, genomic DNA as template and suitable oligonucleotideprimers. The nucleic acid amplified thus can be cloned into a suitablevector and characterized by means of DNA sequence analysis.Oligonucleotides which correspond to a desaturase nucleotide sequencecan be generated by synthetic standard methods, for example using anautomatic DNA synthesizer.

Homologs of the ω-3-desaturase nucleic acid sequence used, with thesequence SEQ ID NO: 1, mean for example allelic variants with at least60%, preferably at least 70%, more preferably at least 80%, 90% or 95%and even more preferably at least approximately 95%, 96%, 97%, 98%, 99%or more identity or homology with a nucleotide sequence shown in SEQ IDNO: 1 or its homologs, derivatives or analogs or parts thereof.Furthermore, homologs are isolated nucleic acid molecules of anucleotide sequence which hybridize, for example under stringentconditions, with the nucleotide sequence shown in SEQ ID NO: 1 or a partthereof. Allelic variants comprise in particular functional variantswhich can be obtained by deletion, insertion or substitution ofnucleotides from/into the sequence shown in SEQ ID NO: 1, the intentionbeing, however, that the enzyme activity of the resulting proteinssynthesized advantageously being retained for the insertion of one ormore genes. Proteins which still retain the enzymatic activity ofω-3-desaturase, i.e. whose activity is essentially not reduced, meanproteins with at least 10%, preferably 20%, especially preferably 30%,very especially preferably 40% of the original enzyme activity incomparison with the protein encoded by SEQ ID NO: 1. The homology wascalculated over the entire amino acid or nucleic acid sequence region. Aseries of programs based on a variety of algorithms is available to theskilled worker for comparing different sequences. In this context, thealgorithms of Needleman and Wunsch or Smith and Waterman giveparticularly reliable results. To carry out the sequence alignments, theprogram PileUp (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al.,CABIOS, 5 1989: 151-153) or the programs Gap and BestFit [Needleman andWunsch (J. Mol. Biol. 48; 443-453 (1970)) and Smith and Waterman (Adv.Appl. Math. 2; 482-489 (1981))], which are part of the GCG softwarepacket [Genetics Computer Group, 575 Science Drive, Madison, Wis., USA53711 (1991)], were used. The sequence homology values detailed above inpercent were determined using the program GAP over the entire sequenceregion with the following settings: Gap Weight: 50, Length Weight: 3,Average Match: 10.000 and Average Mismatch: 0.000, which, unlessotherwise specified, were always used as standard settings for sequencealignments.

Homologs of SEQ ID NO: 1 also mean for example bacterial, fungal andplant homologs, truncated sequences, single-stranded DNA or RNA of thecoding and noncoding DNA sequence.

Homologs of SEQ ID NO: 1 also mean derivatives such as, for example,promoter variants. The promoters upstream of the nucleotide sequencesstated can be modified by one or more nucleotide substitutions, byinsertion(s) and/or deletion(s), without, however, the functionality oractivity of the promoters being adversely affected. Furthermore, it ispossible that the activity of the promoters is increased by modifyingtheir sequence, or that they are replaced completely by more activepromoters, including those from heterologous organisms.

The abovementioned nucleic acids and protein molecules withω-3-desaturase activity which are involved in the metabolism of lipidsand fatty acids, PUFA cofactors and enzymes or in the transport oflipophilic compounds across membranes are used in the process accordingto the invention for modulating the production of PUFAs in transgenicorganisms, advantageously in plants such as maize, wheat, rye, oats,triticale, rice, barley, soybean, peanut, cotton, Linum species such aslinseed or flax, Brassica species such as oilseed rape, canola andturnip, pepper, sunflower, borage, evening primrose and Tagetes,Solanaceae plants such as potato, tobacco, eggplant and tomato, Viciaspecies, pea, cassaya, alfalfa, bushy plants (coffee, cacao, tea), Salixspecies, trees (oil palm, coconut) and perennial grasses and foddercrops either directly (for example when the overexpression oroptimization of a fatty acid biosynthetic protein has a direct effect onthe yield, production and/or production efficiency of the fatty acidfrom modified organisms) and/or can have an indirect effect whichnevertheless entails an increase in the yield, production and/orproduction efficiency of the PUFAs or a decrease of undesired compounds(for example when the modulation of the metabolism of lipids and fattyacids, cofactors and enzymes results in changes in the yield, productionand/or production efficiency or the composition of the desired compoundswithin the cells which, in turn, can have an effect on the production ofone or more fatty acids).

The combination of a variety of precursor molecules and biosyntheticenzymes leads to the production of different fatty acid molecules, whichhas a major effect on the composition of the lipids sincepolyunsaturated fatty acids (═PUFAs) are incorporated not only simplyinto triacylglycerol but also into membrane lipids.

Boraginaceae, Primulaceae or Linaceae are especially suitable for theproduction of PUFAs, for example stearidonic acid, eicosapentaenoic acidor docosahexaenoic acid. Especially advantageously suitable for theproduction of PUFAs with the nucleic acid sequences according to theinvention, advantageously, as described, in combination with furtherdesaturases and elongases, is flax (Linum usitatissimum).

Lipid synthesis can be divided into two sections: the synthesis of fattyacids and their binding to sn-glycerol-3-phosphate, and the addition ormodification of a polar head group. Usual lipids which are used inmembranes comprise phospholipids, glycolipids, sphingolipids andphosphoglycerides. Fatty acid synthesis starts with the conversion ofacetyl-CoA into malonyl-CoA by acetyl-CoA carboxylase or into acetyl-ACPby acetyl transacylase. After condensation reaction, these two productmolecules together form acetoacetyl-ACP, which is converted via a seriesof condensation, reduction and dehydration reactions so that a saturatedfatty acid molecule with the desired chain length is obtained. Theproduction of the unsaturated fatty acids from these molecules iscatalyzed by specific desaturases, either aerobically by means ofmolecular oxygen or anaerobically (regarding the fatty acid synthesis inmicroorganisms, see F. C. Neidhardt et al. (1996) E. coli andSalmonella. ASM Press: Washington, D.C., p. 612-636 and references citedtherein; Lengeler et al. (Ed.) (1999) Biology of Procaryotes. Thieme:Stuttgart, New York, and the references therein, and Magnuson, K., etal. (1993) Microbiological Reviews 57:522-542 and the referencestherein). To undergo the further elongation steps, the resultingphospholipid-bound fatty acids must then be returned from thephospholipids to the fatty acid CoA ester pool. This is made possible byacyl-CoA:lysophospholipid acyltransferases. Moreover, these enzymes arecapable of transferring the elongated fatty acids from the CoA estersback to the phospholipids. If appropriate, this reaction sequence can befollowed repeatedly.

Examples of precursors for PUFA biosynthesis are oleic acid, linoleicacid and linolenic acid. These C₁₈-carbon fatty acids must be elongatedto C₂₀ and C₂₂ to obtain fatty acids of the eicosa and docosa chaintype. It is possible, with the aid of the ω-3-desaturase used in theprocess, to convert arachidonic acid into eicosapentaenoic acid anddocosapentaenoic acid into docosahexaenoic acid and subsequently to usethem for a variety of purposes in applications in the fields offoodstuffs, feedstuffs, cosmetics or pharmaceuticals. Using theabovementioned enzymes, C₁₈-, C₂₀- and/or C₂₂-fatty acids with at leasttwo, advantageously at least three, four, five or six double bonds inthe fatty acid molecule, preferably C₂₀- or C₂₂-fatty acids withadvantageously four, five or six double bonds in the fatty acidmolecule, can be produced. The desaturation can take place before orafter elongation of the fatty acid in question. This is why the productsof the desaturase activities and the further possible desaturation andelongation lead to preferred PUFAs with a higher degree of desaturation,including a further elongation of C₂₀- to C₂₂-fatty acids. Substrates ofthe desaturase used in the process according to the invention are C₁₈-,C₂₀- or C₂₂-fatty acids such as, for example, linoleic acid, γ-linolenicacid, dihomo-γ-linolenic acid, arachidonic acid, docosatetraenoic acidor docosapentaenoic acid. Preferred substrates are arachidonic acid,docosatetraenoic acid or docosapentaenoic acid. The synthesized C₂₀- orC₂₂-fatty acids with at least two, double bonds in the fatty acid areobtained in the process according to the invention in the form of thefree fatty acid or in the form of its esters, for example in the form ofits glycerides.

The term “glyceride” is understood as meaning glycerol esterified withone, two or three carboxyl radicals (mono-, di- or triglyceride).“Glyceride” is also understood as meaning a mixture of variousglycerides. The glyceride or glyceride mixture can comprise furtheradditions, for example free fatty acids, antioxidants, proteins,carbohydrates, vitamins and/or other substances.

A “glyceride” for the purposes of the process according to the inventionis furthermore understood as meaning derivatives which are derived fromglycerol. In addition to the above-described fatty acid glycerides,these also include glycerophospholipids and glyceroglycolipids.Preferred examples which may be mentioned here are theglycerophospholipids such as lecithin (phosphatidylcholine),cardiolipin, phosphatidylglycerol, phosphatidylserine andalkylacylglycerophospholipids. Furthermore, fatty acids mustsubsequently be transported to various sites of modification andincorporated into the triacylglycerol storage lipid. A further importantstep in lipid synthesis is the transfer of fatty acids onto the polarhead groups, for example by glycerol-fatty-acid acyltransferase (seeFrentzen, 1998, Lipid, 100 (4-5):161-166).

For publications on plant fatty acid biosynthesis, desaturation, thelipid metabolism and the membrane transport of fatty compounds,beta-oxidation, fatty acid modification and cofactors, triacylglycerolstorage and assembly, including the references therein, see thefollowing articles: Kinney, 1997, Genetic Engineering, Ed.: J K Setlow,19:149-166; Ohlrogge and Browse, 1995, Plant Cell 7:957-970; Shanklinand Cahoon, 1998, Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:611-641;Voelker, 1996, Genetic Engineering, Ed.: J K Setlow, 18:111-13;Gerhardt, 1992, Prog. Lipid R. 31:397-417; Gühnemann-Schafer & Kindl,1995, Biochim. Biophys Acta 1256:181-186; Kunau et al., 1995, Prog.Lipid Res. 34:267-342; Stymne et al., 1993, in: Biochemistry andMolecular Biology of Membrane and Storage Lipids of Plants, Ed.: Murataand Somerville, Rockville, American Society of Plant Physiologists,150-158, Murphy & Ross 1998, Plant Journal. 13(1):1-16.

The PUFAs produced in the process comprise a group of molecules whichhigher animals are no longer capable of synthesizing and must thereforetake up, or which higher animals are no longer capable of synthesizingthemselves in sufficient quantity and must therefore take upadditionally, although they can be readily synthesized by otherorganisms such as bacteria; for example, cats are no longer capable ofsynthesizing arachidonic acid.

Phospholipids which are advantageously converted by the ω-3-desaturaseaccording to the invention are to be understood as meaning, for thepurposes of the invention, phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylglyceroland/or phosphatidylinositol, advantageously phosphatidylcholine. Theterms “production” or “productivity” are known in the art and refer tothe concentration of the fermentation product (compounds of the formulaI) formed within a certain period of time and a certain fermentationvolume (for example kg of product per hour per liter). The term“production efficiency” comprises the time required for obtaining acertain amount of product (for example the time required by the cell forestablishing a certain throughput rate of a fine chemical). The term“yield” or “product/carbon yield” is known in the art and comprises theefficiency of the conversion of the carbon source into the product (i.e.the fine chemical). This is usually expressed for example as kg ofproduct per kg of carbon source. By increasing the yield or productionof the compound, the amount of the obtained molecules or of the suitableobtained molecules of this compound in a certain amount of culture isincreased over a specified period. The terms “biosynthesis” or“biosynthetic pathway” are known in the art and comprise the synthesisof a compound, preferably of an organic compound, by a cell startingfrom intermediates, for example in a multistep process which is highlyregulated. The terms “catabolism” or “catabolic pathway” are known inthe art and comprise the cleavage of a compound, preferably of anorganic compound, by a cell to give catabolytes (in more general terms,smaller or less complex molecules), for example in a multistep processwhich is highly regulated. The term “metabolism” is known in the art andcomprises the totality of the biochemical reactions which take place inan organism. Thus, the metabolism of a certain compound (for example themetabolism of a fatty acid) comprises the totality of the biosynthetic,modification and catabolic pathways of this compound in the cell, whichrelate to this compound.

In addition to the ω-3-desaturases shown in SEQ ID NO: 1, the skilledworker recognizes that DNA sequence polymorphisms which lead to changesin the amino acid sequences of the ω-3-desaturase may exist within apopulation. These genetic polymorphisms in the ω-3-desaturase gene mayexist between individuals within one population as the result of naturalvariation. These natural variants usually cause a variance of 1 to 5% inthe nucleotide sequence of the ω-3-desaturase gene. All and sundry ofthese nucleotide variations and resulting amino acid polymorphisms inthe ω-3-desaturase which are the result of natural variation and whichdo not change the functional activity shall be covered by the scope ofthe invention.

Nucleic acids molecules which are advantageous for the process accordingto the invention can be isolated on the basis of their homology with theω-3-desaturase nucleic acids disclosed herein, using the sequences orpart thereof as hybridization probe in accordance with standardhybridization techniques under stringent hybridization conditions. Inthis context, for example, it is possible to use isolated nucleic acidmolecules which have a length of at least 15 nucleotides and whichhybridize under stringent conditions with the nucleic acid moleculeswhich comprise a nucleotide sequence of SEQ ID NO: 1. Nucleic acidswhich have at least 25, 50, 100, 250 or more nucleotides may also beused. The term “hybridizes under stringent conditions” as used in thepresent context is intended to describe hybridization and washconditions under which nucleotide sequences which have at least 60%homology with one another usually remain hybridized with one another.The conditions are preferably such that sequences which have at least65%, more preferably at least approximately 70% and even more preferablyat least approximately 75% or more homology with one another usuallyremain hybridized with one another. These stringent conditions are knownto the skilled worker and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred,nonlimiting example for stringent hybridization conditions arehybridization conditions in 6× sodium chloride/sodium citrate (═SSC) atapproximately 45° C., followed by one or more wash steps in 0.2×SSC,0.1% SDS at 50 to 65° C. The skilled worker knows that thesehybridization conditions differ depending on the type of nucleic acidand, for example when organic solvents are present, with regard to thetemperature and concentration of the buffer. For example, under“standard hybridization conditions” the temperature differs depending onthe type of nucleic acid between 42° C. and 58° C. in aqueous bufferwith a concentration of 0.1 to 5×SSC (pH 7.2). If organic solvent ispresent in the abovementioned buffer, for example 50% formamide, thetemperature under standard conditions is approximately 42° C. Thehybridization conditions for DNA:DNA hybrids are preferably for example0.1×SSC and 20° C. to 45° C., preferably between 30° C. and 45° C. Thehybridization conditions for DNA:RNA hybrids are preferably, forexample, 0.1×SSC and 30° C. to 55° C., preferably between 45° C. and 55°C. The abovementioned hybridization temperatures are determined forexample for a nucleic acid with approximately 100 by (=base pairs) inlength and a G+C content of 50% in the absence of formamide. The skilledworker knows how to determine the hybridization conditions required byreferring to textbooks such as the textbook mentioned above, or thefollowing textbooks: Sambrook et al., “Molecular Cloning”, Cold SpringHarbor Laboratory, 1989; Hames and Higgins (Ed.) 1985, “Nucleic AcidsHybridization: A Practical Approach”, IRL Press at Oxford UniversityPress, Oxford; Brown (Ed.) 1991, “Essential Molecular Biology: APractical Approach”, IRL Press at Oxford University Press, Oxford.

To determine the percentage homology (=identity) of two amino acidsequences (for example the sequence SEQ ID NO: 2) or of two nucleicacids (for example SEQ ID NO: 1), the sequences are written one underthe other for the purposes of optimal comparison (for example, gaps maybe introduced into the sequence of a protein or of a nucleic acid inorder to bring about an optimal alignment with the other protein or theother nucleic acid). The amino acid residues or nucleotides at thecorresponding amino acid positions or nucleotide positions are thencompared. If a position in one sequence is occupied by the same aminoacid residue or the same nucleotide as the corresponding position in theother sequence, the molecules are homologous at this position (i.e,amino acid or nucleic acid “homology” as used in the present contextcorresponds to amino acid or nucleic acid “identity”). The percentagehomology between the two sequences is a function of the number ofidentical positions which the sequences share (i.e. % homology=number ofidentical positions/total number of positions×100). The terms homologyand identity can thus be regarded as being synonymous. The programs andalgorithms used are described above.

An isolated nucleic acid molecule which codes for an ω-3-desaturase andwhich is homologous to the protein sequence of SEQ ID NO: 2 can begenerated by introducing one or more nucleotide substitutions, additionsor deletions into a nucleotide sequence of SEQ ID NO: 1, so that one ormore amino acid substitutions, addition or deletions are introduced intothe encoded protein. Mutations can be introduced into one of thesequence of SEQ ID NO: 1 by standard techniques, such as site-specificmutagenesis and PCR-mediated mutagenesis. It is preferred to generateconservative amino acid substitutions at one or more of the predictednonessential amino acid residues. In a “conservative amino acidsubstitution”, the amino acid residue is exchanged for an amino acidresidue with a similar side chain. Families of amino acid residues withsimilar side chains have been defined in the art. These familiescomprise amino acids with basic side chains (for example lysine,arginine, histidine), acidic side chains (for example aspartic acid,glutamic acid), uncharged polar side chains (for example glycine,asparagine, glutamine, serine, threonine, tyrosine, cysteine), unpolarside chains (for example alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan), beta-branched side chains (forexample threonine, valine, isoleucine) and aromatic side chains (forexample tyrosine, phenylalanine, tryptophan, histidine). A predictednonessential amino acid residue in a lysophosphatidic acidacyltransferase, glycerol-3-phosphate acyltransferase, diacylglycerolacyltransferase or lecithin cholesterol acyltransferase is thuspreferably exchanged for another amino acid residue from the sameside-chain family. As an alternative, in another embodiment, themutations can be introduced randomly over the entire sequence coding forlysophosphatic acid acyltransferase, glycerol-3-phosphateacyltransferase, diacylglycerol acyltransferase or lecithin cholesterolacyltransferase or a part of this sequence, for example by saturationmutagenesis, and the resultant mutants can be screened for thelysophosphatidic acid acyltransferase, glycerol-3-phosphateacyltransferase, diacylglycerol acyltransferase or lecithin cholesterolacyltransferase activity described here, in order to identify mutantswhich have retained the lysophosphatidic acid acyltransferase,glycerol-3-phosphate acyltransferase, diacylglycerol acyltransferase orlecithin cholesterol acyltransferase activity. After the mutagenesis ofone of the sequences of SEQ ID NO: 1, the protein encoded can beexpressed recombinantly, and the activity of the protein can bedetermined for example using the assays described herein.

The invention furthermore relates to transgenic nonhuman organisms whichcomprise the nucleic acids SEQ ID NO: 1 according to the invention or agene construct or a vector which comprise these nucleic acid sequencesaccording to the invention. The nonhuman organism is preferably amicroorganism, a nonhuman animal or a plant, especially preferably aplant.

This invention is illustrated in greater detail by the examples whichfollow, which are not to be construed as limiting. The content of all ofthe references, patent applications, patents and published patentapplications cited in the present patent application is herewithincorporated by reference.

EXAMPLES Example 1 General Cloning Methods

The cloning methods such as, for example, restriction cleavages, agarosegel electrophoresis, purification of DNA fragments, transfer of nucleicacids to nitrocellulose and nylon membranes, linkage of DNA fragments,transformation of Escherichia coli cells, bacterial cultures and thesequence analysis of recombinant DNA were carried out as described bySambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN0-87969-309-6).

Example 2 Sequence Analysis of Recombinant DNA

Recombinant DNA molecules were sequenced with an ABI laser fluorescenceDNA sequencer by the process of Sanger (Sanger et al. (1977) Proc. Natl.Acad. Sci. USA 74, 5463-5467). Fragments resulting from a polymerasechain reaction were sequenced and verified to avoid polymerase errors inconstructs to be expressed.

Example 3 Cloning the ω-3-Specific Desaturase from Phytophthorainfestans

As the result of a search for conserved regions in the protein sequencescorresponding to the desaturase genes detailed in the description, onesequence with suitable motifs characteristic of a desaturase wasidentified in an EST sequence database.

Name of gene Genbank No. Amino acids Pi-omega3Des SEQ ID NO: 1 361

Total RNA from Phytophthora infestans was isolated with the aid of theRNAeasy Kit from Qiagen (Valencia, Calif., US) and used for establishingan EST sequence database. Poly-A+ RNA (mRNA) was isolated from the totalRNA with the aid of oligo-dT cellulose (Sambrook et al., 1989). The RNAwas subjected to reverse transcription using the reverse transcriptionsystem kit from Promega, and the cDNA synthesized was cloned into thelambda ZAP vector (lambda ZAP Gold, Stratagene). The cDNA was depackagedin accordance with the manufacturer's instructions to give the plasmidDNA. The cDNA plasmid library was then used for the PCR for cloningexpression plasmids. The sequence data were deposited in a suitabledatabase.

Example 4 Cloning an Expression Plasmid to Express the Pi-Omega3DesHeterologously in Yeast

To express the Pi-omega3Des clone heterologously in yeast, the formerwas cloned by PCR into the yeast expression vector pYES3, using suitablePi-omega3Des-specific primers. In doing so, exclusively the gene's openreading frame which codes for the Pi-omega3Des protein was amplified andprovided with two cleavage sites for cloning into the expression vectorpYES3:

Forward Primer: 5′-TAAGCTTACATGGCGACGAAGGAGG (SEQ ID NO: 3) ReversePrimer: 5′-TGGATCCACTTACGTGGACTTGGT (SEQ ID NO: 4)Composition of the PCR mixture (50 μl):5.00 μl template cDNA5.00 μl 10× buffer (Advantage polymerase)+25 mM MgCl₂5.00 μl 2 mM dNTP1.25 μl per primer (10 pmol/μl of the 5′-ATG and of the 3′-stop primer)0.50 μl Advantage polymeraseThe Advantage polymerase employed was from Clontech.

PCR Reaction Conditions:

Annealing temperature: 1 min 55° C.Denaturation temperature: 1 min 94° C.Elongation temperature: 2 min 72° C.Number of cycles: 35

The PCR product was incubated for 2 hours at 37° C. with the restrictionenzymes HindIII and BamHI. The yeast expression vector pYES3(Invitrogen) was incubated in the same manner. Thereafter, the 1104 byPCR product and the vector were separated by agarose gel electrophoresisand the corresponding DNA fragments were excised. The DNA was purifiedby means of the Qiagen Gel Purification Kit following the manufacturer'sinstructions. Thereafter, the vector and the desaturase cDNA wereligated. The Rapid Ligation Kit from Roche was used for this purpose.The resulting plasmid pYES3-Pi-omega3Des was verified by sequencing andtransformed into the Saccharomyces strain INVSc1 (Invitrogen) by meansof electroporation (1500 V). As a control, pYES3 was transformed inparallel. Thereafter, the yeasts were plated onto complete tryptophanminimal medium with 2% glucose. Cells which were capable of growing evenwithout tryptophan in the medium thus comprise the correspondingplasmids pYES3, pYES3-Pi-omega3Des. After the selection, in each casetwo transformants were selected for the further functional expression.

Example 5 Cloning Expression Plasmids for the Seed-Specific Expressionin Plants

To transform plants, a further transformation vector based on pSUN-USPwas generated. To this end, NotI cleavage sites were introduced at the5′ and 3′ termini of the coding sequence using the following primerpair:

pSUN-Pi-Omega3Des

Reverse: 3′-GCGGCCGCTTACGTGGACTTGGTC (SEQ ID NO: 5) Forward:5′-GCGGCCGCatGGCGACGAAGGAGG (SEQ ID NO: 6)Composition of the PCR mixture (50 μl):5.00 μl template cDNA5.00 μl 10× buffer (Advantage polymerase)+25 mM MgCl₂5.00 μl 2 mM dNTP1.25 μl per primer (10 pmol/μl)0.50 μl Advantage polymeraseThe Advantage polymerase employed was from Clontech.

PCR Reaction Conditions:

Annealing temperature: 1 min 55° C.Denaturation temperature: 1 min 94° C.Elongation temperature: 2 min 72° C.Number of cycles: 35

The PCR products were incubated with the restriction enzyme NotI for 4hours at 37° C. The plant expression vector pSUN300-USP was incubated inthe same manner. Thereafter, the PCR products and the 7624 by vectorwere separated by agarose gel electrophoresis, and the corresponding DNAfragments were excised. The DNA was purified by means of the Qiagen GelPurification Kit following the manufacturer's instructions. Thereafter,vector and PCR products were ligated. The Rapid Ligation Kit from Rochewas used for this purpose. The resulting plasmid pSUN-Pi-omega3Des wasverified by sequencing.

Example 6 Expression of Pi-Omega3Des in Yeasts

Yeasts which had been transformed with the plasmid pYES3 orpYES3-Pi-omega3Des as described in Example 4 were analyzed as follows:

The yeast cells from the main cultures were harvested by centrifugation(100×g, 5 min, 20° C.) and washed with 100 mM NaHCO₃, pH 8.0 in order toremove a residual medium and fatty acids. Fatty acid methyl esters(FAMEs) were prepared from the yeast cell sediments by acidmethanolysis. To this end, the cell sediments were incubated for 1 hourat 80° C. with 2 ml of 1 N methanolic sulfuric acid and 2% (v/v)dimethoxypropane. The FAMEs were extracted by twice extracting withpetroleum ether (PE). To remove non-derivatized fatty acids, the organicphases were washed in each case once with 2 ml of 100 mM NaHCO₃, pH 8.0,and 2 ml of distilled water. Thereafter, the PE phases were dried withNa₂SO₄, evaporated under argon and taken up in 100 μl of PE. The sampleswere separated on a DB-23 capillary column (30 m, 0.25 mm, 0.25 μm,Agilent) in a Hewlett-Packard 6850 gas chromatograph with flameionization detector. The conditions for the GLC analysis were asfollows: the oven temperature was programmed from 50° C. to 250° C. withan increment of 5° C./min and finally 10 minutes at 250° C. (holding).The signals were identified by comparing the retention times withcorresponding fatty acid standards (Sigma). The methodology is describedfor example in Napier and Michaelson, 2001, Lipids 36(8):761-766;Sayanova et al., 2001, Journal of Experimental Botany,52(360):1581-1585, Sperling et al., 2001, Arch. Biochem. Biophys.388(2):293-298 and Michaelson et al., 1998, FEBS Letters.439(3):215-218.

Example 7 Functional Characterization of Pi-Omega3Des

The substrate specificity was determined after expression and feedingwith various fatty acids (FIGS. 2 to 8). The fed substrates are presentin large amounts in all transgenic yeasts, which proves the uptake ofthese fatty acids into the yeasts. The transgenic yeasts show that newfatty acids have been synthesized, the products of the Pi-omega3Desreaction. This means that the gene Pi-omega3Des was expressedfunctionally.

FIG. 2 shows the desaturation of linoleic acid (18:2 ω-6-fatty acid) toα-linolenic acid (18:3 ω-3-fatty acid) by Pi-omega3Des. The fatty acidmethyl esters are synthesized by subjecting intact cells which had beentransformed with the blank vector pYES2 (FIG. 2 A) or the vectorpYes3-Pi-omega3Des (FIG. 2 B) to acid methanolysis. The yeasts weregrown in minimal medium in the presence of C18:2^(Δ9,12)-fatty acid (300μM). The FAMEs were then analyzed via GLC.

FIG. 3 shows the desaturation of γ-linolenic acid (18:3 ω-6-fatty acid)to stearidonic acid (18:4 ω-3-fatty acid) by Pi-omega3Des. The fattyacid methyl esters are synthesized by subjecting intact cells which hadbeen transformed with the blank vector pYES2 (FIG. 3 A) or the vectorpYes3-Pi-omega3Des (FIG. 3 B) to acid methanolysis. The yeasts weregrown in minimal medium in the presence of γ-C18:3^(Δ6,9,12)-fatty acid(300 μM). The FAMEs were then analyzed via GLC.

FIG. 4 shows the desaturation of C20:2 ω-6-fatty acid to C20:3 ω-3-fattyacid by Pi-omega3Des. The fatty acid methyl esters are synthesized bysubjecting intact cells which had been transformed with the blank vectorpYES2 (FIG. 4 A) or the vector pYes3-Pi-omega3Des (FIG. 4 B) to acidmethanolysis. The yeasts were grown in minimal medium in the presence ofC20:2^(Δ11,14)-fatty acid (300 μM). The FAMEs were then analyzed viaGLC.

FIG. 5 shows the desaturation of C20:3 ω-6-fatty acid to C20:4 ω-3-fattyacid by Pi-omega3Des. The fatty acid methyl esters are synthesized bysubjecting intact cells which had been transformed with the blank vectorpYES2 (FIG. 5 A) or the vector pYes3-Pi-omega3Des (FIG. 5 B) to acidmethanolysis. The yeasts were grown in minimal medium in the presence ofC20:3^(Δ8,11,14)-fatty acid (300 μM). The FAMEs were then analyzed viaGLC.

FIG. 6 shows the desaturation of arachidonic acid (C20:4 ω-6-fatty acid)to eicosapentaenoic acid (C20:5 ω-3-fatty acid) by Pi-omega3Des. Thefatty acid methyl esters are synthesized by subjecting intact cellswhich had been transformed with the blank vector pYES2 (FIG. 6 A) or thevector pYes3-Pi-omega3Des (FIG. 6 B) to acid methanolysis. The yeastswere grown in minimal medium in the presence of C20:4^(Δ5,8,11,14)-fattyacid (300 μM). The FAMEs were then analyzed via GLC.

FIG. 7 shows the desaturation of docosatetraenoic acid (C22:4 ω-6-fattyacid) to docosapentaenoic acid (C22:5 ω-3-fatty acid) by Pi-omega3Des.The fatty acid methyl esters are synthesized by subjecting intact cellswhich had been transformed with the blank vector pYES2 (FIG. 7 A) or thevector pYes3-Pi-omega3Des (FIG. 7 B) to acid methanolysis. The yeastswere grown in minimal medium in the presence ofC22:4^(Δ7,10,13,16)-fatty acid (300 μM). The FAMEs were then analyzedvia GLC.

The substrate specificity of Pi-omega3Des with regard to various fattyacids can be seen from FIG. 8. The yeasts which have been transformedwith the vector pYes3-Pi-omega3Des were grown in minimal medium in thepresence of the fatty acids stated. The fatty acid methyl esters weresynthesized by subjecting intact cells to acid methanolysis. The FAMEswere subsequently analyzed by GLC. Each value represents a mean fromthree measurements. The conversion rates (% desaturation) werecalculated using the formula:

[product]/[product]+[substrate]*100.

Example 8 Lipid Extraction from Seeds

The effect of the genetic modification in plants, fungi, algae, ciliatesor on the production of a desired compound (such as a fatty acid) can bedetermined by growing the modified microorganisms or the modified plantunder suitable conditions (such as those described above) and analyzingthe medium and/or the cellular components for the elevated production ofthe desired product (i.e. of the lipids or a fatty acid). Theseanalytical techniques are known to the skilled worker and comprisespectroscopy, thin-layer chromatography, various types of stainingmethods, enzymatic and microbiological methods and analyticalchromatography such as high-performance liquid chromatography (see, forexample, Ullman, Encyclopedia of Industrial Chemistry, Vol. A2, p. 89-90and p. 443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987)“Applications of HPLC in Biochemistry” in: Laboratory Techniques inBiochemistry and Molecular Biology, Vol. 17; Rehm et al. (1993)Biotechnology, Vol. 3, Chapter III: “Product recovery and purification”,p. 469-714, VCH: Weinheim; Better, P. A., et al. (1988) Bioseparations:downstream processing for Biotechnology, John Wiley and Sons; Kennedy,J. F., and Cabral, J. M. S. (1992) Recovery processes for biologicalMaterials, John Wiley and Sons; Shaeiwitz, J. A., and Henry, J. D.(1988) Biochemical Separations, in: Ullmann's Encyclopedia of IndustrialChemistry, Vol. B3; Chapter 11, p. 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, NoyesPublications).

In addition to the abovementioned methods, plant lipids are extractedfrom plant material as described by Cahoon et al. (1999) Proc. Natl.Acad. Sci. USA 96 (22):12935-12940 and Browse et al. (1986) AnalyticBiochemistry 152:141-145. The qualitative and quantitative analysis oflipids or fatty acids is described by Christie, William W., Advances inLipid Methodology, Ayr/Scotland: Oily Press (Oily Press Lipid Library;2); Christie, William W., Gas Chromatography and Lipids. A PracticalGuide—Ayr, Scotland: Oily Press, 1989, Repr. 1992, IX, 307 pp. (OilyPress Lipid Library; 1); “Progress in Lipid Research, Oxford: PergamonPress, 1 (1952)-16 (1977) under the title: Progress in the Chemistry ofFats and Other Lipids CODEN.

In addition to measuring the end product of the fermentation, it is alsopossible to analyze other components of the metabolic pathways which areused for the production of the desired compound, such as intermediatesand by-products, in order to determine the overall production efficiencyof the compound. The analytical methods comprise measuring the amount ofnutrients in the medium (for example sugars, hydrocarbons, nitrogensources, phosphate and other ions), measuring the biomass compositionand the growth, analyzing the production of conventional metabolites ofbiosynthetic pathways and measuring gases which are generated during thefermentation. Standard methods for these measurements are described inApplied Microbial Physiology; A Practical Approach, P. M. Rhodes and P.F. Stanbury, Ed., IRL Press, p. 103-129; 131-163 and 165-192 (ISBN:0199635773) and references cited therein.

One example is the analysis of fatty acids (abbreviations: FAME, fattyacid methyl ester; GC-MS, gas liquid chromatography/mass spectrometry;TAG, triacylglycerol; TLC, thin-layer chromatography).

The unambiguous detection for the presence of fatty acid products can beobtained by analyzing recombinant organisms using analytical standardmethods: GC, GC-MS or TLC, as described on several occasions by Christieand the references therein (1997, in: Advances on Lipid Methodology,Fourth Edition: Christie, Oily Press, Dundee, 119-169; 1998,Gaschromatographie-Massenspektrometrie-Verfahren [Gaschromatography/mass spectrometry methods], Lipide 33:343-353).

The material to be analyzed can be disrupted by sonication, grinding ina glass mill, liquid nitrogen and grinding or via other applicablemethods. After disruption, the material must be centrifuged. Thesediment is resuspended in distilled water, heated for 10 minutes at100° C., cooled on ice and recentrifuged, followed by extraction for onehour at 90° C. in 0.5 M sulfuric acid in methanol with 2%dimethoxypropane, which leads to hydrolyzed oil and lipid compounds,which give transmethylated lipids. These fatty acid methyl esters areextracted in petroleum ether and finally subjected to a GC analysisusing a capillary column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25m, 0.32 mm) at a temperature gradient of between 170° C. and 240° C. for20 minutes and 5 minutes at 240° C. The identity of the resulting fattyacid methyl esters must be defined using standards which are availablefrom commercial sources (i.e. Sigma).

Plant material is initially homogenized mechanically by comminuting in apestle and mortar to make it more amenable to extraction.

This is followed by heating at 100° C. for 10 minutes and, after coolingon ice, by resedimentation. The cell sediment is hydrolyzed for one hourat 90° C. with 1 M methanolic sulfuric acid and 2% dimethoxypropane, andthe lipids are transmethylated. The resulting fatty acid methyl esters(FAMEs) are extracted in petroleum ether. The extracted FAMEs areanalyzed by gas liquid chromatography using a capillary column(Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 m, 0.32 mm) and atemperature gradient of from 170° C. to 240° C. in 20 minutes and 5minutes at 240° C. The identity of the fatty acid methyl esters isconfirmed by comparison with corresponding FAME standards (Sigma). Theidentity and position of the double bond can be analyzed further bysuitable chemical derivatization of the FAME mixtures, for example togive 4,4-dimethoxyoxazolin derivatives (Christie, 1998) by means ofGC-MS.

Example 9 Generation of Transgenic Plants a) Generation of TransgenicOilseed Rape Plants (Modified Process of Moloney et al., 1992, PlantCell Reports, 8:238-242)

Binary vectors in Agrobacterium tumefaciens C58C1:pGV2260 or Escherichiacoli (Deblaere et al., 1984, Nucl. Acids. Res. 13, 4777-4788) were usedfor generating transgenic oilseed rape plants. To transform oilseed rapeplants (Var. Drakkar, NPZ Nordeutsche Pflanzenzucht, Hohenlieth,Germany), a 1:50 dilution of an overnight culture of a positivelytransformed agrobacterial colony in Murashige-Skoog medium (Murashigeand Skoog 1962 Physiol. Plant. 15, 473) supplemented with 3% sucrose(3MS medium) was used. Petiols or hypocotyls of freshly germinatedsterile oilseed rape plants (in each case approx. 1 cm²) were incubatedwith a 1:50 agrobacterial dilution for 5-10 minutes in a petri dish.This is followed by 3 days of coincubation in the dark at 25° C. on 3MSmedium supplemented with 0.8% Bacto agar. The cultures were then grownfor 3 days at 16 hours light/8 hours dark. The cultivation is thencontinued in a weekly rhythm on MS medium supplemented with 500 mg/lClaforan (cefotaxime sodium), 50 mg/l kanamycin, 20 μM benzylaminopurine(BAP) and 1.6 g/l of glucose. Growing shoots were transferred to MSmedium supplemented with 2% sucrose, 250 mg/l Claforan and 0.8% Bactoagar. If no roots had developed after three weeks, 2-indolebutyric acidwas added to the medium as growth hormone for rooting.

Regenerated shoots were obtained on 2MS medium supplemented withkanamycin and Claforan; after rooting, they were transferred to compostand, after growing for two weeks in a controlled-environment cabinet orin the greenhouse, allowed to flower, and mature seeds were harvestedand analyzed by lipid analyses for ω-3-desaturase expression. In thismanner, lines with elevated contents of polyunsaturated C₂₀- andC₂₂-fatty acids were identified.

b) Generation of Transgenic Linseed Plants

Transgenic linseed plants can be generated for example by the process ofBell et al., 1999, In Vitro Cell. Dev. Biol.-Plant. 35(6):456-465 bymeans of particle bombardment. Agrobacteria-mediated transformations canbe effected for example by the process of Mlynarova et al. (1994), PlantCell Report 13: 282-285.

EQUIVALENTS

Many equivalents of the specific embodiments according to the inventiondescribed herein can be seen or found by the skilled worker by simpleroutine experiments. These equivalents are intended to be included inthe patent claims.

1. An isolated nucleic acid molecule comprising a nucleic acid sequencecoding for a polypeptide with ω-3-desaturase activity selected from thegroup consisting of: a) the nucleic acid sequence of SEQ ID NO: 1, b) anucleic acid sequence coding for the amino acid sequence of SEQ ID NO:2, c) a nucleic acid sequence which, as the result of the degeneracy ofthe genetic code, can be derived from the amino acid sequence of SEQ IDNO: 2, d) a nucleic acid sequence having at least 60% identity with thenucleic acid sequence of SEQ ID NO: 1 and coding for a polypeptide withω-3-desaturase activity, and e) a nucleic acid sequence coding for apolypeptide which has at least 60% identity with the amino acid sequenceof SEQ ID NO: 2 and has ω-3-desaturase activity.
 2. The isolated nucleicacid molecule of claim 1, wherein the nucleic acid sequence is derivedfrom an alga, a fungus, a microorganism or a nonhuman animal.
 3. Theisolated nucleic acid molecule of claim 1, wherein the nucleic acidsequence is derived from the family Pythiaceae.
 4. The isolated nucleicacid molecule of claim 1, wherein the nucleic acid sequence has at least95% identity with the nucleic acid sequence of SEQ ID NO: 1 and codesfor a polypeptide with ω-3-desaturase activity.
 5. The isolated nucleicacid molecule of claim 1, wherein the nucleic acid sequence codes for apolypeptide having at least 95% identity with the amino acid sequence ofSEQ ID NO: 2 and having ω-3-desaturase activity.
 6. A gene constructcomprising the isolated nucleic acid molecule of claim 1, wherein thenucleic acid sequence is linked functionally to one or more regulatorysignals.
 7. The gene construct of claim 6, wherein the gene constructcomprises additional biosynthesis genes of fatty acid or lipidmetabolism selected from the group consisting of acyl-CoAdehydrogenase(s), acyl-ACP [=acyl carrier protein] desaturase(s),acyl-ACP thioesterase(s), fatty acid acyltransferase(s),acyl-CoA:lysophospholipid acyltransferase(s), fatty acid synthase(s),fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s),acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acidacetylenases, lipoxygenases, triacylglycerol lipases, allenoxidesynthases, hydroperoxide lyases and fatty acid elongase(s).
 8. The geneconstruct of claim 6, wherein the gene construct additionally comprisesbiosynthesis genes of fatty acid or lipid metabolism selected from thegroup consisting of Δ4-desaturase, Δ5-desaturase, Δ6-desaturase,Δ8-desaturase, Δ9-desaturase, Δ12-desaturase, Δ6-elongase, Δ5-elongaseand Δ9-elongase.
 9. A vector comprising the nucleic acid molecule ofclaim 1 or a gene construct comprising said nucleic acid moleculefunctionally linked to one or more regulatory signals.
 10. A transgenicnonhuman organism comprising at least one nucleic acid molecule of claim1, a gene construct comprising said at least one nucleic acid moleculefunctionally linked to one or more regulatory signals, or a vectorcomprising said at least one nucleic acid molecule or said geneconstruct.
 11. The transgenic nonhuman organism of claim 10, wherein theorganism is a microorganism, a nonhuman animal, or a plant.
 12. Thetransgenic nonhuman organism of claim 10, wherein the organism is aplant.
 13. The transgenic nonhuman organism of claim 10, wherein theorganism is an oil-producing plant, a vegetable producing plant, or anornamental plant.
 14. The transgenic nonhuman organism of claim 10,wherein the organism is a plant selected from the group of plantfamilies consisting of Adelotheciaceae, Anacardiaceae, Asteraceae,Apiaceae, Betulaceae, Boraginaceae, Brassicaceae, Bromeliaceae,Caricaceae, Cannabaceae, Convolvulaceae, Chenopodiaceae,Crypthecodiniaceae, Cucurbitaceae, Ditrichaceae, Elaeagnaceae,Ericaceae, Euphorbiaceae, Fabaceae, Geraniaceae, Gramineae,Juglandaceae, Lauraceae, Leguminosae, Linaceae and Prasinophyceae.